BR122024018518A2 - METHOD FOR SELECTION OF M6P-RICH RECOMBINANT PROTEINS - Google Patents

Abstract

Methods for the production, capture and purification of recombinant human lysosomal proteins are described. Such recombinant human lysosomal proteins may have a high content of mannose-6-phosphate residues. Pharmaceutical compositions comprising such recombinant human lysosomal proteins are also described, as are methods of treatment and uses of such recombinant human lysosomal proteins.

Description

TECHNICAL AREA

[0001] The principles and embodiments of the present invention relate to the manufacture of recombinant proteins, particularly lysosomal enzymes that have a high mannose-6-phosphate content.

BACKGROUND

[0002] Lysosomal storage disorders are a group of autosomal recessive genetic diseases characterized by the accumulation of cellular glucosphingolipids, glycogen, or mucopolysaccharides within intracellular compartments called lysosomes. Individuals with these diseases carry mutant genes encoding enzymes that are defective in catalyzing the hydrolysis of one or more of these substances, which then accumulate in lysosomes. For example, Pompe disease, also known as acid maltase deficiency or glycogen storage disease type II, is one of several lysosomal storage disorders. Other examples of lysosomal disorders include Gaucher disease, GM1-gangliosidosis, fucosidosis, mucopolysaccharidoses, Hurler-Scheie disease, Niemann-Pick diseases A and B, and Fabry disease. Pompe disease is also classified as a neuromuscular disease or a metabolic myopathy.

[0003] Pompe disease is estimated to occur in about 1 in 40,000 births and is caused by a mutation in the GAA gene, which encodes the enzyme lysosomal α-glucosidase (EC:3.2.1.20), also commonly known as acid α-glucosidase. Acid α-glucosidase is involved in the metabolism of glycogen, a branched polysaccharide that is the major storage form of glucose in animals, by catalyzing its hydrolysis to glucose within lysosomes. Because individuals with Pompe disease produce defective, mutant acid α-glucosidase that is inactive or has reduced activity, glycogen breakdown occurs slowly or does not occur at all, and glycogen accumulates in the lysosomes of various tissues, particularly striated muscle, leading to a wide spectrum of clinical manifestations, including progressive muscle weakness and respiratory failure. Tissues such as cardiac and skeletal muscles are particularly affected.

[0004] Pompe disease can vary widely in the degree of enzyme deficiency, severity, and age of onset, and more than 500 different mutations in the GAA gene have been identified, many of which cause disease symptoms of varying severity. The disease has been classified into broad types: early-onset or infantile-onset and late-onset. Early-onset disease and lower enzyme activity are generally associated with a more severe clinical course. Infantile Pompe disease is the most severe, resulting from complete or near-complete deficiency of acid α-glucosidase, and presents with symptoms that include severe muscle tone, weakness, enlarged liver and heart, and cardiomyopathy. The tongue may become broad and protruding, and swallowing may become difficult. Most affected children die of respiratory or cardiac complications before the age of two years. Late-onset Pompe disease can present at any age above 12 months and is characterized by a lack of cardiac involvement and a better short-term prognosis. Symptoms are related to progressive skeletal muscle dysfunction and involve generalized muscle weakness and wasting of respiratory muscles in the trunk, proximal lower limbs, and diaphragm. Some adult patients are free of significant symptoms or motor limitations. Prognosis generally depends on the extent of respiratory muscle involvement. Most individuals with Pompe disease eventually progress to physical debilitation requiring the use of a wheelchair and assisted ventilation, with premature death often occurring due to respiratory failure.

[0005] Recent treatment options for Pompe disease include enzyme replacement therapy (ERT) with recombinant human acid α-glucosidase (rhGAA). Conventional rhGAA products are known under the names alglucosidase alfa, Myozyme® or Lumizyme® from Genzyme, Inc. ERT is a chronic treatment required throughout the patient's life and involves administration of the replacement enzyme by intravenous infusion. The replacement enzyme is then transported into the circulation and enters lysosomes within cells, where it acts to break down accumulated glycogen, compensating for the deficient activity of the endogenous defective mutant enzyme and thus alleviating the symptoms of the disease. In subjects with infantile-onset Pompe disease, treatment with alglucosidase alfa has been shown to significantly improve survival compared with historical controls, and in late-onset Pompe disease, alglucosidase alfa has been shown to have a statistically significant, if modest, effect on the 6-Minute Walk Test (6MWT) and forced vital capacity (FVC) compared with placebo.

[0006] However, most subjects remain stable or continue to deteriorate while undergoing alglucosidase alfa treatment. The reason for the apparent suboptimal effect of ERT with alglucosidase alfa is unclear, but could be in part due to the progressive nature of the underlying muscle pathology or the poor tissue targeting of current ERT. For example, the infused enzyme is not stable at neutral pH, including at plasma pH (approximately pH 7.4), and may be irreversibly inactivated within the circulation. Furthermore, infused alglucosidase alfa shows insufficient uptake into key disease-relevant muscles, possibly due to inadequate glycosylation with mannose-6-phosphate (M6P) residues. Such residues bind cation-independent mannose-6-phosphate receptors (CIMPR) on the cell surface, allowing the enzyme to enter the cell and internal lysosomes. Therefore, high doses of the enzyme may be required for effective treatment so that an adequate amount of active enzyme can reach the lysosomes, making therapy expensive and time-consuming.

[0007] There are seven potential N-linked glycosylation sites on rhGAA. Since each glycosylation site is heterogeneous in the type of N-linked oligosaccharides (N-glycans) present, rhGAA consists of a complex mixture of proteins with N-glycans having varying binding affinities for the M6P receptor and other carbohydrate receptors. rhGAA containing a high-mannose N-glycan bearing one M6P group (mono-M6P) binds to CIMPR with low affinity (~6,000 nM) while rhGAA containing two M6P groups on the same N-glycan (bis-M6P) binds with high affinity (~2 nM). Representative structures for unphosphorylated glycans, mono-M6P and bis-M6P are shown in Figure 1A. The mannose-6-P group is shown in Figure 1B. Once inside the lysosome, rhGAA can enzymatically degrade accumulated glycogen. However, conventional rhGAAs have low total levels of glycans carrying M6P and bis-M6P and thus poorly target muscle cells resulting in inferior distribution of rhGAA to lysosomes. Productive drug targeting of rhGAA is shown in Figure 2A. Most of the rhGAA molecules in these conventional products lack phosphorylated N-glycans and thus lack affinity for CIMPR. Unphosphorylated high-mannose glycans can also be cleared by the mannose receptor resulting in nonproductive clearance of ERT (Figure 2B).

[0008] The other type of N-glycans, complex carbohydrates, which contain galactose and sialic acids, are also present on rhGAA. Since complex N-glycans are not phosphorylated they have no affinity for CIMPR. However, complex-type N-glycans with exposed galactose residues have a moderate to high affinity for the asialoglycoprotein receptor on liver hepatocytes, which leads to rapid nonproductive clearance of rhGAA (Figure 2B).

[0009] Current manufacturing processes used to manufacture conventional rhGAA, such as Myozyme®, Lumizyme® or alglucosidase alfa, did not significantly increase the content of M6P or bis-M6P because cellular processing of carbohydrates is inherently complex and extremely difficult to manipulate. Accordingly, there remains a need for further improvements to enzyme replacement therapy for the treatment of Pompe disease, such as new manufacturing, capture and purification processes for rhGAA.

[0010] Similarly, other recombinant proteins that are targeted to the lysosome, such as other lysosomal enzymes, also bind to CIMPR. However, current manufacturing processes used to manufacture other conventional recombinant proteins that are targeted to lysosomes do not provide recombinant proteins with a high content of M6P or bis-M6P. Accordingly, there remains a need for further improvements in the manufacturing, capture and purification processes for these other recombinant proteins as well. SUMMARY

[0011] One aspect of the present invention relates to a method for producing recombinant human lysosomal proteins. In various embodiments of this aspect, the method comprises culturing host cells in a bioreactor that secrete a recombinant human lysosomal protein, removing media from the bioreactor, filtering the media to provide a filtrate, loading the filtrate onto an anion exchange chromatography (AEX) column to capture the lysosomal protein, and eluting a first protein product from the AEX column.

[0012] In certain embodiments, the recombinant human lysosomal protein is recombinant human α-glucosidase (rhGAA). In certain embodiments, the rhGAA comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 2.

[0013] In one or more embodiments, the method further comprises loading the first protein product onto a chromatography column and eluting a second protein product from the column. In some embodiments, the column is an immobilized metal affinity chromatography (IMAC) column.

[0014] In one or more embodiments, the method further comprises loading the second protein product onto a chromatography column and eluting a third protein product from the chromatography column. In some embodiments, the third chromatography column is selected from a cation exchange chromatography (CEX) column and a size exclusion chromatography (SEC) column.

[0015] In one or more embodiments, the filtration of the media is selected from alternating tangential flow filtration (ATF) and tangential flow filtration (TFF).

[0016] In one or more embodiments, the method further comprises inactivating viruses in one or more of the first protein product, the second protein product, and the third protein product.

[0017] In one or more embodiments, the method further comprises filtering the second protein product or the third protein product to provide a filtered product and filling a vial with the filtered product. In one or more embodiments, the method further comprises lyophilizing the filtered product.

[0018] In one or more embodiments, the host cells comprise Chinese hamster ovary (CHO) cells. In one or more embodiments, the host cells comprise the CHO cell line GA-ATB-200 or ATB-200-X5-14 or a subculture thereof.

[0019] In certain embodiments, (i) at least 90% of the first protein product or the second protein product or the third protein product binds to the cation-independent mannose-6-phosphate receptor (CIMPR), or (ii) at least 90% of the first protein product or the second protein product or the third protein product contains an N-glycan carrying mono-mannose-6-phosphate (mono-M6P) or bis-mannose-6-phosphate (bis-M6P).

[0020] In certain embodiments, the recombinant human lysosomal protein is rhGAA comprising seven potential N-glycosylation sites, at least 50% of rhGAA molecules comprise an N-glycan unit carrying two mannose-6-phosphate residues at the first site, at least 30% of rhGAA molecules comprise an N-glycan unit carrying one mannose-6-phosphate residue at the second site, at least 30% of rhGAA molecules comprise an N-glycan unit carrying two mannose-6-phosphate residues at the fourth site, and at least 20% of rhGAA molecules comprise an N-glycan unit carrying one mannose-6-phosphate residue at the fourth site.

[0021] Another aspect of the present invention relates to a recombinant protein product prepared by any of the methods described herein.

[0022] Another aspect of the present invention relates to a pharmaceutical composition comprising the recombinant protein product and a pharmaceutically acceptable carrier.

[0023] Yet another aspect of the present invention relates to a method for treating a lysosomal storage disorder, the method comprising administering the pharmaceutical composition to a patient in need thereof.

[0024] In certain embodiments, the lysosomal storage disorder is Pompe disease and the recombinant protein is rhGAA. In certain embodiments, a pharmacological chaperone for α-glucosidase is co-administered to the patient within 4 hours of administration of the pharmaceutical composition comprising the rhGAA product. In some embodiments, the pharmacological chaperone is selected from 1-deoxynojirimycin and N-butyl-deoxynojirimycin. In some embodiments, the pharmacological chaperone is co-formulated with the rhGAA product.

[0025] Various embodiments are listed below. It will be understood that the embodiments listed below may be combined not only as listed below, but in other suitable combinations in accordance with the scope of the invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Additional features of the present invention will become apparent from the following written description and accompanying figures, in which:

[0027] Figure 1A shows unphosphorylated high-mannose glycan, a mono-M6P glycan, and a bis-M6P glycan.

[0028] Figure 1B shows the chemical structure of the M6P group.

[0029] Figure 2A depicts the productive targeting of rhGAA via M6P-carrying glycans to target tissues (e.g., muscle tissues from subjects with Pompe Disease).

[0030] Figure 2B depicts non-productive clearance of drug to non-target tissues (e.g., liver and spleen) or by binding of non-M6P glycans to non-target tissues.

[0031] Figure 3A graphically illustrates a CIMPR receptor (also known as an IGF2 receptor) and domains of this receptor.

[0032] Figure 3B is a table showing the binding affinity (nmolar) of bis- and mono-M6P-bearing glycans for CIMPR, the binding affinity of high-mannose-type glycans for mannose receptors, and the binding affinity of desialylated complex glycan for asialioglycoprotein receptors. rhGAA having both M6P- and bis-M6P-bearing glycans can productively bind to CIMPR in muscle.

[0033] Figures 4A and 4B, respectively, are graphs showing the affinity chromatography results for CIMPR from Lumizyme® and Myozyme®. The dashed lines refer to the M6P elution gradient. Elution with M6P displaces GAA molecules bound via an M6P glycan to CIMPR. As shown in Figure 2A, 78% of the GAA activity in Lumizyme® eluted prior to the addition of M6P. Figure 2B shows that 73% of the GAA activity in Myozyme® eluted prior to the addition of M6P. Only 22% or 27% of the rhGAA in Lumizyme® or Myozyme®, respectively, was eluted with M6P. These values show that the majority of rhGAA in these two conventional rhGAA products lack M6P-bearing glycans required to target CIMPR in target muscle tissues.

[0034] Figure 5 shows a DNA construct for transformation of CHO cells with DNA encoding rhGAA. CHO cells were transformed with a DNA construct encoding rhGAA.

[0035] Figure 6 is a schematic diagram of an exemplary process for the manufacture, capture, and purification of a recombinant lysosomal protein.

[0036] Figures 7A and 7B, respectively, are graphs showing the results of affinity chromatography for CIMPR of Myozyme® and rhGAA ATB200. As apparent from Figure 7B, approximately 70% of the rhGAA in rhGAA ATB200 contained M6P.

[0037] Figure 8 is a graph showing the results of CIMPR affinity chromatography of rhGAA ATB200 with and without capture on an anion exchange (AEX) column.

[0038] Figure 9 is a graph showing Polywax elution profiles of Lumizyme® and rhGAA ATB200s.

[0039] Figure 10 is a table showing a summary of the N-glycan structures of Lumizyme® in comparison to three different preparations of rhGAA ATB200, identified as BP-rhGAA, ATB200-1, and ATB200-2.

[0040] Figures 11A-11H show the results of a site-specific N-glycosylation analysis of rhGAA ATB200.

[0041] Figure 12A is a graph comparing the CIMPR binding affinity of rhGAA ATB200 (left trace) with that of Lumizyme® (right trace).

[0042] Figure 12B is a table comparing the Bis-M6P content of Lumizyme® and rhGAA ATB200.

[0043] Figure 13A is a graph comparing rhGAA ATB200 activity (left trace) with Lumizyme® rhGAA activity (right trace) within normal fibroblasts at various GAA concentrations.

[0044] Figure 13B is a table comparing the activity of rhGAA ATB200 (left trace) with the activity of Lumizyme® rhGAA (right trace) within fibroblasts from a subject having Pompe Disease at various concentrations of GAA.

[0045] Figure 13C is a table comparing the Kuptake of fibroblasts from normal subjects and subjects with Pompe Disease.

[0046] Figure 14A is a graph showing the amount of glycogen relative to the dose of recombinant human acid α-glucosidase in mouse heart muscle after contact with vehicle (negative control), with alglucosidase alfa at 20 mg/mL (Lumizyme®) or with ATB200 at 5, 10 or 20 mg/kg.

[0047] Figure 14B is a graph showing the amount of glycogen relative to the dose of recombinant human acid α-glucosidase in mouse quadriceps muscle after contact with vehicle (negative control), with alglucosidase alfa at 20 mg/mL (Lumizyme®) or with ATB200 at 5, 10 or 20 mg/kg.

[0048] Figure 14C is a graph showing the amount of glycogen relative to the dose of recombinant human acid α-glucosidase in mouse triceps muscle after contact with vehicle (negative control), with alglucosidase alfa at 20 mg/mL (Lumizyme®) or with ATB200 at 5, 10 or 20 mg/kg.

[0049] Figure 15 is a table showing that the combination of rhGAA ATB200 and the chaperone miglustat provided significantly better glycogen clearance in GAA knockout mice than treatments with Lumizyme® or ATB200 without the chaperone miglustat.

[0050] Figure 16 is a series of electron micrographs of cardiac, diaphragm, and soleus muscle from wild-type and Gaa knockout mice treated with vehicle, alglucosidase alfa, and ATB200 in the presence and absence of miglustat, showing levels of lysosome-associated membrane protein (LAMP-1).

[0051] Figure 17 is a series of electron micrographs of cardiac and soleus muscle from wild-type and Gaa knockout mice treated with vehicle, alglucosidase alfa, and ATB200 in the presence and absence of miglustat, showing glycogen levels by periodic acid-Schiff (PAS) reagent staining.

[0052] Figure 18 is a series of electron micrographs (1000x) of quadriceps muscle from wild-type and Gaa knockout mice treated with vehicle, alglucosidase alfa, and ATB200 in the presence and absence of miglustat, stained with methylene blue to show vacuoles (indicated by arrows).

[0053] Figure 19 is a series of electron micrographs (40x) of quadriceps muscle from wild-type and Gaa knockout mice treated with vehicle, alglucosidase alfa, and ATB200 in the presence and absence of miglustat, showing levels of the autophagy markers phosphatidylethanolamine conjugate of microtubule-associated protein 1A/1B light chain 3 (LC3A II) and p62, the insulin-dependent glucose transporter GLUT4, and the insulin-independent glucose transporter GLUT1.

[0054] Figures 20A and 20B, respectively, are graphs showing the results of CIMPR affinity chromatography of recombinant α-galactosidase A (rhα-Gal A) enzyme prior to or after capture and purification on an anion exchange (AEX) column.

[0055] Figures 21A and 21B are graphs showing hand muscle fiber and grip strength data for wild-type and Gaa knockout mice treated with vehicle, alglucosidase alfa, and ATB200 in the presence of miglustat.

[0056] Figures 22A-22G are graphs showing glycogen levels in quadriceps, triceps, and cardiac cells from wild-type and Gaa knockout mice treated with vehicle, alglucosidase alfa, and ATB200 in the presence and absence of miglustat.

[0057] Figure 23 is a series of photomicrographs (100x and 200x) of vastus lateralis (VL) muscle fibers from wild-type and Gaa knockout mice treated with vehicle, alglucosidase alfa, and ATB200 in the presence and absence of miglustat, showing signs of dystrophin.

[0058] Figure 24 shows the study design of a Phase 1/2, first-in-human, ascending dose, fixed-sequence, open-label study to evaluate the safety, tolerability, PK, PD, and efficacy of intravenous infusions of ATB200 co-administered with oral miglustat in adults with Pompe disease.

[0059] Figures 25A-25B are graphs showing the concentration-time profiles of total GAA protein in plasma in human subjects following dosing with ATB200 at 5, 10, or 20 mg/kg, ATB200 at 20 mg/kg and 130 mg miglustat, or ATB200 at 20 mg/kg and 260 mg miglustat.

[0060] Figure 25C is a graph showing the AUC of total GAA protein in plasma in human subjects following dosing with ATB200 at 20 mg/kg, ATB200 at 20 mg/kg and 130 mg miglustat, or ATB200 at 20 mg/kg and 260 mg miglustat.

[0061] Figure 25D is a graph showing the concentration-time profiles of total GAA protein in plasma in two individual human subjects following dosing with ATB200 at 20 mg/kg or 260 mg miglustat.

[0062] Figure 26 is a graph showing the plasma concentration-time profiles of miglustat in human subjects following dosing with 130 mg or 260 mg of miglustat.

[0063] Figures 27A-27D are graphs showing changes in alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatine phosphokinase (CPK), and hexose tetrasaccharide (Hex4) levels in human patients following administration of ascending doses of ATB200 (5, 10, and 20 mg/kg) followed by coadministration of ATB200 (20 mg/kg) and miglustat (130 and 260 mg).

DETAILED DESCRIPTION

[0064] Prior to describing various exemplary embodiments of the invention it is to be understood that the invention is not limited to the construction details or process steps set forth in the following description. The invention is capable of other embodiments and of being practiced or carried out in various ways.

[0065] Although specific reference is made to GAA it will be understood by one of skill in the art that the methods described herein can be used to produce, capture and purify other recombinant proteins that target the lysosome, including but not limited to the lysosomal enzyme α-galactosidase A.

[0066] Various aspects of the invention relate to novel methods for the production, capture and purification of recombinant human lysosomal proteins, such as recombinant human acid α-glucosidase (rhGAA). Other aspects of the invention relate to recombinant proteins produced by the processes described herein, as well as pharmaceutical compositions, methods of treatment and uses of such recombinant proteins.

Definitions

[0067] The terms used in this specification generally have their usual meanings in the art within the context of the present invention and in the specific context in which each term is used. Certain terms are discussed below or elsewhere in the specification to provide additional guidance to the practitioner in describing the compositions and methods of the invention and how to make and use the same.

[0068] In the present specification, unless the context otherwise requires due to express language or necessary implication, the word “comprise”, or variations such as “comprises” or “comprising”, is used in an inclusive sense, i.e., to specify the presence of the features shown, but not to preclude the presence or addition of additional features in various embodiments of the invention.

[0069] As used herein, the term “lysosomal protein” refers to any protein that is targeted to the lysosome, such as a lysosomal enzyme. Examples of lysosomal enzymes and associated disease are provided in Table 1 below: Table 1

[0070] As used herein, the term “Pompe disease,” also referred to as acid maltase deficiency or glycogen storage disease type II (GSDII) and glucogenosis type II, is intended to refer to a genetic lysosomal storage disorder characterized by mutations in the GAA gene, which encodes the human acid α-glucosidase enzyme. The term includes, but is not limited to, early- and late-onset forms of the disease, including but not limited to infantile-, juvenile-, and adult-onset Pompe disease.

[0071] As used herein, the term “acid α-glucosidase” is intended to refer to a lysosomal enzyme that hydrolyzes α-1,4 bonds between the D-glucose units of glycogen, maltose, and isomaltose. Alternative names include, but are not limited to, lysosomal α-glucosidase (EC:3.2.1.20); glucoamylase; 1,4-α-D-glucan glucohydrolase; amyloglucosidase; gamma-amylase, and exo-1,4-α-glucosidase. Human acid α-glucosidase is encoded by the GAA gene (National Centre for Biotechnology Information (NCBI) Gene ID 2548), which has been mapped to the long arm of chromosome 17 (location 17q25.2-q25.3). More than 500 mutations have currently been identified in the human GAA gene, many of which are associated with Pompe disease. Mutations resulting in misfolding or incorrect processing of the acid α-glucosidase enzyme include T1064C (Leu355Pro) and C2104T (Arg702Cys). Additionally, GAA mutations that affect maturation and processing of the enzyme include Leu405Pro and Met519Thr. The conserved hexapeptide WIDMNE at amino acid residues 516–521 is required for acid α-glucosidase protein activity. As used herein, the abbreviation “GAA” is intended to refer to the acid α-glucosidase enzyme, while the italicized abbreviation “GAA” is intended to refer to the human gene encoding the human acid α-glucosidase enzyme. The italicized abbreviation “Gaa” is intended to refer to non-human genes encoding non-human acid α-glucosidase enzymes, including but not limited to rat or mouse genes, and the abbreviation “Gaa” is intended to refer to non-human acid α-glucosidase enzymes. Thus, the abbreviation “rhGAA” is intended to refer to the recombinant human acid α-glucosidase enzyme.

[0072] As used herein, the term “alglucosidase alfa” is intended to refer to a recombinant human acid α-glucosidase identified as [199-arginine,223-histidine]prepro-α-glucosidase (human); Chemical Abstracts Registry Number 420794-05-0. Alglucosidase alfa is approved for marketing in the United States by Genzyme as of January 2016 as the products Lumizyme® and Myozyme®.

[0073] As used herein, the term “ATB200” is intended to refer to a recombinant human acid α-glucosidase described in copending patent application PCT/US2015/053252, the disclosure of which is incorporated herein by reference.

[0074] As used herein, the term “glycan” is intended to refer to a polysaccharide chain covalently attached to an amino acid residue in a protein or polypeptide. As used herein, the term “N-glycan” or “N-linked glycan” is intended to refer to a polysaccharide chain attached to an amino acid residue in a protein or polypeptide via covalent bonding to a nitrogen atom of the amino acid residue. For example, an N-glycan may be covalently attached to the nitrogen atom of the side chain of an asparagine residue. Glycans may contain one or more monosaccharide units, and the monosaccharide units may be covalently linked to form a linear chain or a branched chain. In at least one embodiment, the N-glycan units attached to ATB200 may comprise one or more monosaccharide units each independently selected from N-acetylglucosamine, mannose, galactose, or sialic acid. The N-glycan units on the protein can be determined by any appropriate analytical technique, such as mass spectrometry. In some embodiments, the N-glycan units can be determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS) using an instrument such as the Thermo Scientific Orbitrap Velos ProTM Mass Spectrometer, the Thermo Scientific Orbitrap Fusion Lumos TribidTM Mass Spectrometer, or the Waters Xevo® G2-XS QTof Mass Spectrometer.

[0075] As used herein, the term “high-mannose N-glycan” is intended to refer to an N-glycan having one to six or more mannose units. In at least one embodiment, a high-mannose N-glycan unit may contain a bis(N-acetylglucosamine) chain attached to an asparagine residue and further attached to a branched polymannose chain. As used interchangeably herein, the term “M6P” or “mannose-6-phosphate” is intended to refer to a mannose unit phosphorylated at the 6-position; i.e., having a phosphate group attached to the hydroxyl group at the 6-position. In at least one embodiment, one or more mannose units of one or more N-glycan units are phosphorylated at the 6-position to form mannose-6-phosphate units. In at least one embodiment, the term “M6P” or “mannose-6-phosphate” refers to both a mannose phosphodiester having N-acetylglucosamine (GlcNAc) as a “cap” on the phosphate group, as well as a mannose moiety having an exposed phosphate group lacking the GlcNAc cap. In at least one embodiment, the N-glycans of a protein can have multiple M6P groups, with at least one M6P group having a GlcNAc cap and at least one other M6P group lacking a GlcNAc cap.

[0076] As used herein, the term “complex N-glycan” is intended to refer to an N-glycan containing one or more galactose and/or sialic acid units.

[0077] As used herein, the compound miglustat, also known as N-butyl-1-deoxynojirimycin NB-DNJ or (2R,3R,4R,5S)-1-butyl-2-(hydroxymethyl)piperidine-3,4,5-triol, is a compound having the following chemical formula:

[0078] A formulation of miglustat is commercially marketed under the registered name Zavesca® as monotherapy for type 1 Gaucher disease.

[0079] As discussed below, pharmaceutically acceptable salts of miglustat may also be used in the present invention. When a salt of miglustat is used, the dosage of the salt will be adjusted such that the dose of miglustat received by the patient is equivalent to the amount that would have been received if miglustat free base had been used.

[0080] As used herein, the compound divoglustat, also known as 1-deoxynojirimycin or DNJ or (2R,3R,4R,5S)-2-(hydroxymethyl)piperidine-3,4,5-triol, is a compound having the following chemical formula:

[0081] When a duvoglustat salt is used, the dosage of the salt will be adjusted such that the dose of duvoglustat received by the patient is equivalent to the amount that would have been received if duvoglustat free base had been used.

[0082] As used herein, the term “pharmacological chaperone” or sometimes simply the term “chaperone” is intended to refer to a molecule that specifically binds to a lysosomal protein and has one or more of the following effects: enhances the formation of a stable molecular conformation of the protein; enhances the proper trafficking of the protein from the endoplasmic reticulum to another cellular location, preferably a native cellular location, so as to prevent endoplasmic reticulum-associated degradation of the protein; prevents the aggregation of conformationally unstable or misfolded proteins; restores and/or enhances, at least partially, the wild-type function, stability and/or activity of the protein; and/or improves the phenotype or function of the cell harboring the protein.

[0083] Thus, a pharmacological chaperone for acid α-glucosidase is a molecule that binds to acid α-glucosidase, resulting in proper folding, trafficking, non-aggregation, and activity of acid α-glucosidase. As used herein, this term includes, but is not limited to, active site-specific chaperones (ASSCs) that bind at the active site of the enzyme, inhibitors or antagonists, and agonists. In at least one embodiment, the pharmacological chaperone can be an inhibitor or antagonist of acid α-glucosidase. As used herein, the term “antagonist” is intended to refer to any molecule that binds to acid α-glucosidase and that partially or completely blocks, inhibits, reduces, or neutralizes an activity of acid α-glucosidase. In at least one embodiment, the pharmacological chaperone is miglustat. Another non-limiting example of a pharmacological chaperone for acid α-glucosidase is duvoglustat.

[0084] As used herein, the term “active site” is intended to refer to a region of a protein that is associated with and is necessary for a specific biological activity of the protein. In at least one embodiment, the active site can be a site that binds a substrate or other binding partner and contributes amino acid residues that directly participate in the making and breaking of chemical bonds.

[0085] As used herein, “therapeutically effective dose” and “effective amount” are intended to refer to an amount of recombinant human lysosomal protein (e.g., rhGAA) and/or chaperone and/or a combination thereof, that is sufficient to result in a therapeutic response in a subject. A therapeutic response can be any response that a user (e.g., a clinician) will recognize as an effective response to therapy, including any surrogate clinical markers or symptoms described herein and known in the art. Thus, in at least one embodiment, a therapeutic response can be an amelioration or inhibition of one or more symptoms or markers of Pompe disease such as those known in the art. Symptoms or markers of Pompe disease include, but are not limited to, decreased tissue acid α-glucosidase activity; cardiomyopathy; cardiomegaly; progressive muscle weakness, especially in the trunk or lower limbs; profound hypotonia; macroglossia (and in some cases, tongue protrusion); difficulty swallowing, sucking and/or feeding; respiratory failure; hepatomegaly (moderate); facial muscle laxity; areflexia; exercise intolerance; exercise-related dyspnea; orthopnea; sleep apnea; morning headaches; drowsiness; lordosis and/or scoliosis; decreased deep tendon reflexes; low back pain; and failure to meet developmental motor milestones. It should be noted that a concentration of chaperone (e.g., miglustat) that has an inhibitory effect on acid α-glucosidase may constitute an “effective amount” for purposes of this invention due to dilution (and consequent shift in binding due to shift in equilibrium), bioavailability and metabolism of the chaperone following in vivo administration.

[0086] As used herein, the term “enzyme replacement therapy” or “ERT” is intended to refer to the introduction of a purified, non-native enzyme into an individual having a deficiency in such an enzyme. The administered protein may be obtained from natural sources or by recombinant expression. The term also refers to the introduction of a purified enzyme into an individual requiring or otherwise benefiting from the administration of a purified enzyme. In at least one embodiment, such an individual suffers from enzyme insufficiency. The introduced enzyme may be a purified, recombinant enzyme produced in vitro or a protein purified from isolated tissue or fluid, such as, for example, placenta or animal milk or from plants.

[0087] As used herein, the term “combination therapy” is intended to refer to any therapy in which two or more individual therapies are administered simultaneously or consecutively. In at least one embodiment, the results of the combination therapy are enhanced compared to the effect of each therapy when administered individually. Enhancement may include any improvement in the effect of the multiple therapies that may result in an advantageous outcome compared to the results achieved by the therapies when administered alone. Enhanced effect or results may include a synergistic enhancement, wherein the enhanced effect is more than the additive effects of each therapy when administered alone; an additive enhancement, wherein the enhanced effect is substantially equal to the additive effect of each therapy when administered alone; or less than a synergistic effect, wherein the enhanced effect is lower than the additive effect of each therapy when administered alone, but still better than the effect of each therapy when administered alone. The enhanced effect may be measured by any means known in the art by which the efficacy or outcome of the treatment can be measured.

[0088] As used herein, the term “pharmaceutically acceptable” is intended to refer to molecular entities and compositions that are physiologically tolerable and that typically do not generate undesirable reactions when administered to a human. Preferably, as used herein, the term “pharmaceutically acceptable” means approved by a federal or state government regulatory agency or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals and more particularly in humans.

[0089] As used herein, the term “carrier” is intended to refer to a diluent, adjuvant, excipient, or vehicle with which a compound is administered. Suitable pharmaceutical carriers are known in the art and, in at least one embodiment, are described in E. W. Martin's “Remington's Pharmaceutical Sciences,” 18th Edition, or other editions.

[0090] As used herein, the terms “subject” or “patient” are intended to refer to a human or non-human animal. In at least one embodiment, the subject is a mammal. In at least one embodiment, the subject is a human.

[0091] As used herein, the term “anti-drug antibody” is intended to refer to an antibody specifically binding to a drug administered to a subject and generated by the subject as at least part of a humoral immune response to administration of the drug to the subject. In at least one embodiment, the drug is a therapeutic protein drug product. The presence of the anti-drug antibody in the subject may cause immune responses ranging from mild to severe, including but not limited to life-threatening immune responses that include but are not limited to anaphylaxis, cytokine release syndrome, and neutralization of cross-reactivity of endogenous proteins mediating critical functions. Additionally or alternatively, the presence of the anti-drug antibody in the subject may decrease the efficacy of the drug.

[0092] As used herein, the term “neutralizing antibody” is intended to refer to an anti-drug antibody acting to neutralize the function of the drug. In at least one embodiment, the therapeutic protein drug product is a counterpart of an endogenous protein for which expression is reduced or absent in the subject. In at least one embodiment, the neutralizing antibody can act to neutralize the function of the endogenous protein.

[0093] As used herein, the terms “about” and “approximately” are intended to refer to an acceptable degree of error for the quantity measured given the nature or precision of the measurements. For example, the degree of error may be indicated by the number of significant figures provided for the measurement, as is understood in the art, and includes but is not limited to a variation of ±1 in the most precise significant number reported for the measurement. Typical exemplary degrees of error are within 20 percent (%), preferably within 10%, and more preferably within 5% of a certain value or range of values. Alternatively, and particularly in biological systems, the terms “about” and “approximately” may mean values that are within an order of magnitude, preferably within 5 times, and most preferably within 2 times of a given value. Numerical quantities given here are approximate unless otherwise stated, meaning that the term “about” or “approximately” may be inferred when not expressly stated.

[0094] The term “simultaneously” as used herein is intended to mean at the same time or within a reasonably short period of time before or after, as will be understood by those skilled in the art. For example, if two treatments are to be administered simultaneously with each other, one treatment may be administered before or after the other treatment, to allow time to prepare for the second of the two treatments. Therefore, “simultaneous administration” of two treatments includes but is not limited to one treatment following the other by 20 minutes or less, about 20 minutes, about 15 minutes, about 10 minutes, about 5 minutes, about 2 minutes, about 1 minute, or less than 1 minute.

[0095] The term “pharmaceutically acceptable salt” as used herein is intended to mean a salt which is, within the scope of sound medical judgment, suitable for use in contact with tissues of humans and lower animals without undue toxicity, irritation, allergic response and the like, proportionate to a reasonable risk/benefit ratio, generally soluble or dispersible in water or oil and effective for its intended use. The term includes pharmaceutically acceptable acid addition salts and pharmaceutically acceptable base addition salts. Lists of suitable salts are found in, e.g., S. M. Birge et al., J. Pharm. Sci., 1977, 66, pp. 1-19, incorporated herein by reference.

[0096] The term “pharmaceutically acceptable acid addition salt” as used herein is intended to mean those salts which retain the biological effectiveness and properties of the free bases and which are not biologically or otherwise undesirable, formed with inorganic acids including but not limited to hydrochloric acid, hydrobromic acid, sulfuric acid, sulfamic acid, nitric acid, phosphoric acid and the like, and organic acids including but not limited to acetic acid, trifluoroacetic acid, adipic acid, ascorbic acid, aspartic acid, benzenesulfonic acid, benzoic acid, butyric acid, camphoric acid, camphorsulfonic acid, cinnamic acid, citric acid, digluconic acid, ethanesulfonic acid, glutamic acid, glycolic acid, glycerophosphoric acid, hemisulfic acid, hexanoic acid, formic acid, fumaric acid, 2-hydroxyethanesulfonic acid (isethionic acid), lactic acid, hydroxymaleic acid, malic acid, malonic acid, mandelic acid, mesitylenesulfonic acid, methanesulfonic acid, naphthalenesulfonic acid, nicotinic acid, 2-naphthalenesulfonic acid, oxalic acid, pamoic acid, pectinic acid, phenylacetic acid, 3-phenylpropionic acid, pivalic acid, propionic acid, pyruvic acid, salicylic acid, stearic acid, succinic acid, sulfanilic acid, tartaric acid, p-toluenesulfonic acid, undecanoic acid and the like.

[0097] The term “pharmaceutically acceptable base addition salt” as used herein is intended to mean those salts which retain the biological effectiveness and properties of the free acids and which are not biologically or otherwise undesirable, formed with inorganic bases including but not limited to ammonia or ammonium hydroxide, carbonate or bicarbonate or a metal cation such as sodium, potassium, lithium, calcium, magnesium, iron, zinc, copper, manganese, aluminum and the like. Salts derived from pharmaceutically acceptable non-toxic organic bases include, but are not limited to, salts of primary, secondary and tertiary amines, quaternary amine compounds, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins such as methylamine, dimethylamine, trimethylamine, ethylamine, diethylamine, triethylamine, isopropylamine, tripropylamine, tributylamine, ethanolamine, diethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, hydrabamine, choline, betaine, ethylenediamine, glucosamine, methylglucamine, theobromine, purines, piperazine, piperidine, N-ethylpiperidine, tetramethylammonium compounds, tetraethylammonium compounds, pyridine, N,N-dimethylaniline, N-methylpiperidine, N-methylmorpholine, dicyclohexylamine, dibenzylamine, N,N-dibenzylphenethylamine, 1-ephenamine, N,N'-dibenzylethylenediamine, polyamine resins and the like.

rhGAA ATB200

[0098] In at least one embodiment, the recombinant human lysosomal protein (e.g., rhGAA) is expressed in Chinese hamster ovary (CHO) cells and comprises an increased content of N-glycan units carrying one or more mannose-6-phosphate residues as compared to a content of N-glycan units carrying one or more mannose-6-phosphate residues of a recombinant human lysosomal protein such as alglucosidase alfa. In at least one embodiment, the acid α-glucosidase is a recombinant human α-glucosidase referred to herein as ATB200 as described in copending international patent application PCT/US2015/053252. ATB200 has been shown to bind to cation-independent mannose-6-phosphate receptors (CIMPR) with high affinity (KD ~ 2-4 nM) and is efficiently internalized by Pompe fibroblasts and skeletal muscle myoblasts (Kuptake ~ 7-14 nM). ATB200 has been characterized in vivo and has been shown to have a shorter apparent plasma half-life (t1/2 ~ 45 min) than alglucosidase alfa (t1/2 ~ 60 min).

[0099] In at least one embodiment, the recombinant human acid α-glucosidase is an enzyme having an amino acid sequence as set forth in SEQ ID NO: 1 or SEQ ID NO: 2. SEQ ID NO: 1 Met Gly Val Arg His Pro Pro Cys Ser His Arg Leu Leu Ala Val Cys Ala Leu Val Ser Leu Ala Thr Ala Ala Leu Leu Gly His Ile Leu Leu His Asp Phe Leu Leu Val Pro Arg Glu Leu Ser Gly Ser Pro Val Leu Glu Glu Thr His Pro Ala His Gln Gln Gly Ala Ser Arg Pro Gly Pro Arg Asp Ala Gln Ala His Pro Gly Arg Pro Arg Ala Val Pro Thr Gln Cys Asp Val Pro Pro Asn Ser Arg Phe Asp Cys Ala Pro Asp Lys Ala Ile Thr Gln Glu Gln Cys Glu Ala Arg Gly Cys Cys Tyr Ile Pro Ala Lys Gln Gly Leu Gln Gly Ala Gln Met Gly Gln Pro Trp Cys Phe Phe Pro Pro Ser Tyr Pro Ser Tyr Lys Leu Glu Asn Leu Ser Ser Glu Met Gly Tyr Thr Ala Thr Leu Thr Arg Thr Thr Pro Thr Phe Phe Pro Lys Asp Ile Leu Thr Leu Arg Leu Asp Val Met Met Glu Thr Glu Asn Arg Leu His Phe Thr Ile Lys Asp Pro Ala Asn Arg Arg Tyr Glu Val Pro Leu Glu Thr Pro Arg Val His Ser Arg Ala Pro Ser Pro Leu Tyr Ser Val Glu Phe Ser Glu Glu Pro Phe Gly Val Ile Val His Arg Gln Leu Asp Gly Arg Val Leu Leu Asn Thr Thr Val Ala Pro Leu Phe Phe Ala Asp Gln Phe Leu Gln Leu Ser Thr Ser Leu Pro Ser Gln Tyr Ile Thr Gly Leu Ala Glu His Leu Ser Pro Leu Met Leu Ser Thr Ser Trp Thr Arg Ile Thr Leu Trp Asn Arg Asp Leu Ala Pro Thr Pro Gly Ala Asn Leu Tyr Gly Ser His Pro Phe Tyr Leu Ala Leu Glu Asp Gly Gly Ser Ala His Gly Val Phe Leu Asn Ser Asn Ala Met Asp Val Val Leu Gln Pro Ser Pro Ala Leu Ser Trp Arg Ser Thr Gly Gly Ile Leu Asp Val Tyr Ile Phe Leu Gly Pro Glu Pro Lys Ser Val Val Gln Gln Tyr Leu Asp Val Val Gly Tyr Pro Phe Met Pro Pro Tyr Trp Gly Leu Gly Phe His Leu Cys Arg Trp Gly Tyr Ser Ser Thr Ala Ile Thr Arg Gln Val Val Glu Asn Met Thr Arg Ala His Phe Pro Leu Asp Val Gln Trp Asn Asp Leu Asp Tyr Met Asp Ser Arg Arg Asp Phe Thr Phe Asn Lys Asp Gly Phe Arg Asp Phe Pro Ala Met Val Gln Glu Leu His Gln Gly Gly Arg Arg Tyr Met Met Ile Val Asp Pro Ala Ile Ser Ser Ser Gly Pro Ala Gly Ser Tyr Arg Pro Tyr Asp Glu Gly Leu Arg Arg Gly Val Phe Ile Thr Asn Glu Thr Gly Gln Pro Leu Ile Gly Lys Val Trp Pro Gly Ser Thr Ala Phe Pro Asp Phe Thr Asn Pro Thr Ala Leu Ala Trp Trp Glu Asp Met Val Ala Glu Phe His Asp Gln Val Pro Phe Asp Gly Met Trp Ile Asp Met Asn Glu Pro Ser Asn Phe Ile Arg Gly Ser Glu Asp Gly Cys Pro Asn Asn Glu Leu Glu Asn Pro Pro Tyr Val Pro Gly Val Val Gly Gly Thr Leu Gln Ala Ala Thr Ile Cys Ala Ser Ser His Gln Phe Leu Ser Thr His Tyr Asn Leu His Asn Leu Tyr Gly Leu Thr Glu Ala Ile Ala Ser His Arg Ala Leu Val Lys Ala Arg Gly Thr Arg Pro Phe Val Ile Ser Arg Ser Thr Phe Ala Gly His Gly Arg Tyr Ala Gly His Trp Thr Gly Asp Val Trp Ser Ser Trp Glu Gln Leu Ala Ser Ser Val Pro Glu Ile Leu Gln Phe Asn Leu Leu Gly Val Pro Leu Val Gly Ala Asp Val Cys Gly Phe Leu Gly Asn Thr Ser Glu Glu Leu Cys Val Arg Trp Thr Gln Leu Gly Ala Phe Tyr Pro Phe Met Arg Asn His Asn Ser Leu Leu Ser Leu Pro Gln Glu Pro Tyr Ser Phe Ser Glu Pro Ala Gln Gln Ala Met Arg Lys Ala Leu Thr Leu Arg Tyr Ala Leu Leu Pro His Leu Tyr Thr Leu Phe His Gln Ala His Val Ala Gly Glu Thr Val Ala Arg Pro Leu Phe Leu Glu Phe Pro Lys Asp Ser Ser Thr Trp Thr Val Asp His Gln Leu Leu Trp Gly Glu Ala Leu Leu Ile Thr Pro Val Leu Gln Ala Gly Lys Ala Glu Val Thr Gly Tyr Phe Pro Leu Gly Thr Trp Tyr Asp Leu Gln Thr Val Pro Ile Glu Ala Leu Gly Ser Leu Pro Pro Pro Pro Ala Ala Pro Arg Glu Pro Ala Ile His Ser Glu Gly Gln Trp Val Thr Leu Pro Ala Pro Leu Asp Thr Ile Asn Val His Leu Arg Ala Gly Tyr Ile Ile Pro Leu Gln Gly Pro Gly Leu Thr Thr Thr Glu Ser Arg Gln Gln Pro Met Ala Leu Ala Val Ala Leu Thr Lys Gly Gly Glu Ala Arg Gly Glu Leu Phe Trp Asp Asp Gly Glu Ser Leu Glu Val Leu Glu Arg Gly Ala Tyr Thr Gln Val Ile Phe Leu Ala Arg Asn Asn Thr Ile Val Asn Glu Leu Val Arg Val Thr Ser Glu Gly Ala Gly Leu Gln Leu Gln Lys Val Thr Val Leu Gly Val Ala Thr Ala Pro Gln Gln Val Leu Ser Asn Gly Val Pro Val Ser Asn Phe Thr Tyr Ser Pro Asp Thr Lys Val Leu Asp Ile Cys Val Ser Leu Leu Met Gly Glu Gln Phe Leu Val Ser Trp Cys SEQ ID NO: 2 Gln Gln Gly Ala Ser Arg Pro Gly Pro Arg Asp Ala Gln Ala His Pro Gly Arg Pro Arg Ala Val Pro Thr Gln Cys Asp Val Pro Pro Asn Ser Arg Phe Asp Cys Ala Pro Asp Lys Ala Ile Thr Gln Glu Gln Cys Glu Ala Arg Gly Cys Cys Tyr Ile Pro Ala Lys Gln Gly Leu Gln Gly Ala Gln Met Gly Gln Pro Trp Cys Phe Phe Pro Pro Ser Tyr Pro Ser Tyr Lys Leu Glu Asn Leu Ser Ser Ser Glu Met Gly Tyr Thr Ala Thr Leu Thr Arg Thr Thr Pro Thr Phe Phe Pro Lys Asp Ile Leu Thr Leu Arg Leu Asp Val Met Met Glu Thr Glu Asn Arg Leu His Phe Thr Ile Lys Asp Pro Ala Asn Arg Arg Tyr Glu Val Pro Leu Glu Thr Pro Arg Val His Ser Arg Ala Pro Ser Pro Leu Tyr Ser Val Glu Phe Ser Glu Glu Pro Phe Gly Val Ile Val His Arg Gln Leu Asp Gly Arg Val Leu Leu Asn Thr Thr Val Ala Pro Leu Phe Phe Ala Asp Gln Phe Leu Gln Leu Ser Thr Ser Leu Pro Ser Gln Tyr Ile Thr Gly Leu Ala Glu His Leu Ser Pro Leu Met Leu Ser Thr Ser Trp Thr Arg Ile Thr Leu Trp Asn Arg Asp Leu Ala Pro Thr Pro Gly Ala Asn Leu Tyr Gly Ser His Pro Phe Tyr Leu Ala Leu Glu Asp Gly Gly Ser Ala His Gly Val Phe Leu Leu Asn Ser Asn Ala Met Asp Val Val Leu Gln Pro Ser Pro Ala Leu Ser Trp Arg Ser Thr Gly Gly Ile Leu Asp Val Tyr Ile Phe Leu Gly Pro Glu Pro Lys Ser Val Val Gln Gln Tyr Leu Asp Val Val Gly Tyr Pro Phe Met Pro Pro Tyr Trp Gly Leu Gly Phe His Leu Cys Arg Trp Gly Tyr Ser Ser Thr Ala Ile Thr Arg Gln Val Val Glu Asn Met Thr Arg Ala His Phe Pro Leu Asp Val Gln Trp Asn Asp Leu Asp Tyr Met Asp Ser Arg Arg Asp Phe Thr Phe Asn Lys Asp Gly Phe Arg Asp Phe Pro Ala Met Val Gln Glu Leu His Gln Gly Gly Arg Arg Tyr Met Met Ile Val Asp Pro Ala Ile Ser Ser Ser Gly Pro Ala Gly Ser Tyr Arg Pro Tyr Asp Glu Gly Leu Arg Arg Gly Val Phe Ile Thr Asn Glu Thr Gly Gln Pro Leu Ile Gly Lys Val Trp Pro Gly Ser Thr Ala Phe Pro Asp Phe Thr Asn Pro Thr Ala Leu Ala Trp Trp Glu Asp Met Val Ala Glu Phe His Asp Gln Val Pro Phe Asp Gly Met Trp Ile Asp Met Asn Glu Pro Ser Asn Phe Ile Arg Gly Ser Glu Asp Gly Cys Pro Asn Asn Glu Leu Glu Asn Pro Pro Tyr Val Pro Gly Val Val Gly Gly Thr Leu Gln Ala Ala Thr Ile Cys Ala Ser Ser His Gln Phe Leu Ser Thr His Tyr Asn Leu His Asn Leu Tyr Gly Leu Thr Glu Ala Ile Ala Ser His Arg Ala Leu Val Lys Ala Arg Gly Thr Arg Pro Phe Val Ile Ser Arg Ser Thr Phe Ala Gly His Gly Arg Tyr Ala Gly His Trp Thr Gly Asp Val Trp Ser Ser Trp Glu Gln Leu Ala Ser Ser Val Pro Glu Ile Leu Gln Phe Asn Leu Leu Gly Val Pro Leu Val Gly Ala Asp Val Cys Gly Phe Leu Gly Asn Thr Ser Glu Glu Leu Cys Val Arg Trp Thr Gln Leu Gly Ala Phe Tyr Pro Phe Met Arg Asn His Asn Ser Leu Leu Ser Leu Pro Gln Glu Pro Tyr Ser Phe Ser Glu Pro Ala Gln Gln Ala Met Arg Lys Ala Leu Thr Leu Arg Tyr Ala Leu Leu Pro His Leu Tyr Thr Leu Phe His Gln Ala His Val Ala Gly Glu Thr Val Ala Arg Pro Leu Phe Leu Glu Phe Pro Lys Asp Ser Ser Thr Trp Thr Val Asp His Gln Leu Leu Trp Gly Glu Ala Leu Leu Ile Thr Pro Val Leu Gln Ala Gly Lys Ala Glu Val Thr Gly Tyr Phe Pro Leu Gly Thr Trp Tyr Asp Leu Gln Thr Val Pro Ile Glu Ala Leu Gly Ser Leu Pro Pro Pro Pro Ala Ala Pro Arg Glu Pro Ala Ile His Ser Glu Gly Gln Trp Val Thr Leu Pro Ala Pro Leu Asp Thr Ile Asn Val His Leu Arg Ala Gly Tyr Ile Ile Pro Leu Gln Gly Pro Gly Leu Thr Thr Thr Glu Ser Arg Gln Gln Pro Met Ala Leu Ala Val Ala Leu Thr Lys Gly Gly Glu Ala Arg Gly Glu Leu Phe Trp Asp Asp Gly Glu Ser Leu Glu Val Leu Glu Arg Gly Ala Tyr Thr Gln Val Ile Phe Leu Ala Arg Asn Asn Thr Ile Val Asn Glu Leu Val Arg Val Thr Ser Glu Gly Ala Gly Leu Gln Leu Gln Lys Val Thr Val Leu Gly Val Ala Thr Ala Pro Gln Gln Val Leu Ser Asn Gly Val Pro Val Ser Asn Phe Thr Tyr Ser Pro Asp Thr Lys Val Leu Asp Ile Cys Val Ser Leu Leu Met Gly Glu Gln Phe Leu Val Ser Trp Cys

[0100] In at least one embodiment, the recombinant human acid α-glucosidase has a wild-type GAA amino acid sequence as set forth in SEQ ID NO: 1, as described in U.S. Patent No. 8,592,362 and has GenBank accession number AHE24104.1 (GI:568760974). In at least one embodiment, the recombinant human acid α-glucosidase is glucosidase alpha, the human acid α-glucosidase enzyme encoded by the most predominant of the nine observed haplotypes of the GAA gene.

[0101] In at least one embodiment, the recombinant human acid α-glucosidase is initially expressed as having the full-length 952 amino acid sequence of wild-type GAA as set forth in SEQ ID NO: 1, and the recombinant human acid α-glucosidase is subjected to intracellular processing that removes a portion of the amino acids, e.g., the first 56 amino acids. Accordingly, the recombinant human acid α-glucosidase that is secreted by the host cell may have a shorter amino acid sequence than the recombinant human acid α-glucosidase that is initially expressed within the cell. In at least one embodiment, the shorter protein may have the amino acid sequence set forth in SEQ ID NO: 2, which differs only from SEQ ID NO: 1 in that the first 56 amino acids comprising the signal peptide and precursor peptide have been removed, thereby resulting in a protein having 896 amino acids. Other variations in the number of amino acids are also possible, such as having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more deletions, substitutions and/or insertions relative to the amino acid sequence described by SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, the rhGAA product includes a mixture of recombinant human acid α-glucosidase molecules having different amino acid lengths.

[0102] In at least one embodiment, the recombinant human acid α-glucosidase is subjected to post-translational and/or chemical modifications at one or more amino acid residues in the protein. For example, methionine and tryptophan residues can be subjected to oxidation. As another example, N-terminal glutamine can form pyroglutamate. As another example, asparagine residues can be subjected to deamidation to aspartic acid. As yet another example, aspartic acid residues can undergo isomerization to iso-aspartic acid. As yet another example, unpaired cysteine residues in the protein can form disulfide bonds with free glutathione and/or cysteine. Accordingly, in some embodiments, the enzyme is initially expressed as having an amino acid sequence as set forth in SEQ ID NO: 1 or SEQ ID NO: 2, and the enzyme undergoes one or more of these post-translational and/or chemical modifications. Such modifications are also within the scope of the present disclosure.

[0103] Polynucleotide sequences encoding GAA and such variant human GAAs are also contemplated and may be used to recombinantly express rhGAAs in accordance with the invention.

[0104] Preferably, no more than 70, 65, 60, 55, 45, 40, 35, 30, 25, 20, 15, 10 or 5% of the total recombinant human lysosomal protein (e.g., rhGAA) molecules lack an N-glycan unit carrying one or more mannose-6-phosphate residues or lack the ability to bind to the cation-independent mannose-6-phosphate receptor (CIMPR). Alternatively, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 99%, <100% of recombinant human lysosomal protein molecules (e.g., rhGAA) comprise at least one N-glycan unit carrying one or more mannose-6-phosphate residues or have the ability to bind to CIMPR.

[0105] Recombinant human lysosomal protein molecules (e.g., rhGAA) may have 1, 2, 3, or 4 mannose-6-phosphate (M6P) groups on their glycans. For example, only one N-glycan on a recombinant human lysosomal protein molecule may carry M6P (mono-phosphorylated), a single N-glycan may carry two M6P groups (bis-phosphorylated), or two different N-glycans on the same recombinant human lysosomal protein molecule may each carry unique M6P groups. Recombinant human lysosomal protein molecules may also have N-glycans not carrying M6P groups. In another embodiment, on average, the N-glycans contain greater than 3 mol/mol M6P and greater than 4 mol/mol sialic acid, such that the recombinant human lysosomal protein comprises on average at least 3 moles of mannose-6-phosphate residues per mole of recombinant human lysosomal protein and at least 4 moles of sialic acid per mole of recombinant human lysosomal protein. On average, at least about 3, 4, 5, 6, 7, 8, 9, or 10% of the total glycans on the recombinant human lysosomal protein may be in the form of a mono-M6P glycan, for example, about 6.25% of the total glycans may carry a single M6P group, and on average at least about 0.5, 1, 1.5, 2.0, 2.5, 3.0% of the total glycans on the recombinant human lysosomal protein are in the form of a bis-M6P glycan, and on average less than 25% of the total recombinant human lysosomal protein contains no phosphorylated glycan binding to CIMPR.

[0106] The recombinant human lysosomal protein (e.g., rhGAA) may have an average content of M6P-bearing N-glycans ranging from 0.5 to 7.0 mol/mol lysosomal protein or any intermediate value within the subrange including 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, or 7.0 mol/mol lysosomal protein. The lysosomal protein may be fractionated to provide lysosomal protein preparations with different average numbers of M6P-bearing or bis-M6P-bearing glycans thereby allowing further customization of targeting of the lysosomal protein to lysosomes in target tissues by selection of a particular fraction or by selective combination of different fractions.

[0107] In some embodiments, the recombinant human lysosomal protein (e.g., rhGAA) will carry, on average, 2.0 to 8.0 moles of M6P per mole of recombinant human lysosomal protein (e.g., rhGAA). This range includes all intermediate values and subranges including 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, and 8.0 mol M6P/mol recombinant human lysosomal protein (e.g., rhGAA).

[0108] Up to 60% of the N-glycans on the recombinant human lysosomal protein (e.g., rhGAA) can be fully sialylated, for example, up to 10%, 20%, 30%, 40%, 50%, or 60% of the N-glycans can be fully sialylated. In some embodiments, from 4 to 20% of the total N-glycans are fully sialylated. In other embodiments, no more than 5%, 10%, 20%, or 30% of the N-glycans on the recombinant human lysosomal protein (e.g., rhGAA) carry sialic acid and a terminal galactose (Gal) residue. This range includes all intermediate values and subranges, for example, 7 to 30% of the total N-glycans on the recombinant human lysosomal protein can carry sialic acid and terminal galactose. In still other embodiments, no more than 5, 10, 15, 16, 17, 18, 19, or 20% of the N-glycans on the recombinant human lysosomal protein have only a terminal galactose and do not contain sialic acid. This range includes all intermediate values and subranges, e.g., from 8 to 19% of the total N-glycans on the recombinant human lysosomal protein in the composition may have only terminal galactose and do not contain sialic acid.

[0109] In other embodiments of the invention, 40, 45, 50, 55 to 60% of the total N-glycans on the recombinant human lysosomal protein (e.g., rhGAA) in the composition are complex-type N-glycans; or no more than 1, 2, 3, 4, 5, 6, 7% of the total N-glycans on the recombinant human lysosomal protein (e.g., rhGAA) are hybrid-type N-glycans; no more than 5, 10, or 15% of the high-mannose-type N-glycans on the recombinant human lysosomal protein (e.g., rhGAA) are not phosphorylated; at least 5% or 10% of the high-mannose-type N-glycans on the recombinant human lysosomal protein (e.g., rhGAA) are mono-M6P phosphorylated; and/or at least 1 or 2% of the high-mannose N-glycans on the recombinant human lysosomal protein (e.g., rhGAA) are bis-M6P phosphorylated. These values include all intermediate values and subranges. A recombinant human lysosomal protein (e.g., rhGAA) may correspond to one or more of the contents of the ranges described above.

[0110] In some embodiments, the recombinant human lysosomal protein (e.g., rhGAA) will carry, on average, 2.0 to 8.0 moles of sialic acid residues per mole of recombinant human lysosomal protein (e.g., rhGAA). This range includes all intermediate values and subranges including 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, and 8.0 mole residues/mole recombinant human lysosomal protein (e.g., rhGAA). Without being limited by theory, it is believed that the presence of N-glycan moieties carrying sialic acid residues may prevent nonproductive clearance of the recombinant human lysosomal protein (e.g., rhGAA) by asialoglucoprotein receptors.

[0111] In certain embodiments, the recombinant human lysosomal protein (e.g., rhGAA) has M6P and/or sialic acid units at certain N-glycosylation sites of the recombinant human lysosomal protein. For example, as set forth above, there are seven potential N-linked glycosylation sites in rhGAA. Such potential glycosylation sites are at the following positions of SEQ ID NO: 2: N84, N177, N334, N414, N596, N826, and N869. Similarly, for the full-length amino acid sequence of SEQ ID NO: 1, such potential glycosylation sites are at the following positions: N140, N233, N390, N470, N652, N882, and N925. Other variants of rhGAA may have similar glycosylation sites, depending on the location of the asparagine residues. Generally, the sequences ASN-X-SER or ASN-X-THR in the protein amino acid sequence indicate potential glycosylation sites, with the exception that X cannot be HIS or PRO.

[0112] In various embodiments, rhGAA has a certain N-glycosylation profile. In certain embodiments, at least 20% of the rhGAA is phosphorylated at the first N-glycosylation site (e.g., N84 for SEQ ID NO: 2 and N140 for SEQ ID NO: 1). For example, at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the rhGAA can be phosphorylated at the first N-glycosylation site. This phosphorylation can be the result of mono-M6P and/or bis-M6P units. In some embodiments, at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the rhGAA carries a mono-M6P unit at the first N-glycosylation site. In some embodiments, at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the rhGAA carries a bis-M6P unit at the first N-glycosylation site.

[0113] In certain embodiments, at least 20% of the rhGAA is phosphorylated at the second N-glycosylation site (e.g., N177 for SEQ ID NO: 2 and N223 for SEQ ID NO: 1). For example, at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the rhGAA can be phosphorylated at the second N-glycosylation site. This phosphorylation can be the result of mono-M6P and/or bis-M6P units. In some embodiments, at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the rhGAA carries a mono-M6P unit at the second N-glycosylation site. In some embodiments, at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the rhGAA carries a bis-M6P unit at the second N-glycosylation site. In certain embodiments, at least 5% of the rhGAA is phosphorylated at the third N-glycosylation site (e.g., N334 for SEQ ID NO: 2 and N390 for SEQ ID NO: 1). In other embodiments, less than 5%, 10%, 15%, 20%, or 25% of the rhGAA is phosphorylated at the third N-glycosylation site. For example, the third N-glycosylation site can have a mixture of unphosphorylated high-mannose glycans, two-, three-, or four-antenna complex glycans, and hybrid glycans as the major species. In some embodiments, at least 3%, 5%, 8%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% of the rhGAA is sialylated at the third N-glycosylation site.

[0114] In certain embodiments, at least 20% of the rhGAA is phosphorylated at the fourth N-glycosylation site (e.g., N414 for SEQ ID NO: 2 and N470 for SEQ ID NO: 1). For example, at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the rhGAA can be phosphorylated at the fourth N-glycosylation site. This phosphorylation can be the result of mono-M6P and/or bis-M6P units. In some embodiments, at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the rhGAA carries a mono-M6P unit at the fourth N-glycosylation site. In some embodiments, at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the rhGAA carries a bis-M6P unit at the fourth N-glycosylation site. In some embodiments, at least 3%, 5%, 8%, 10%, 15%, 20%, or 25% of the rhGAA is sialylated at the fourth N-glycosylation site.

[0115] In certain embodiments, at least 5% of the rhGAA is phosphorylated at the fifth N-glycosylation site (e.g., N596 for SEQ ID NO: 2 and N692 for SEQ ID NO: 1). In other embodiments, less than 5%, 10%, 15%, 20%, or 25% of the rhGAA is phosphorylated at the fifth N-glycosylation site. For example, the fifth N-glycosylation site can have fucosylated two-antenna complex glycans as the major species. In some embodiments, at least 3%, 5%, 8%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the rhGAA is sialylated at the fifth N-glycosylation site.

[0116] In certain embodiments, at least 5% of the rhGAA is phosphorylated at the sixth N-glycosylation site (e.g., N826 for SEQ ID NO: 2 and N882 for SEQ ID NO: 1). In other embodiments, less than 5%, 10%, 15%, 20%, or 25% of the rhGAA is phosphorylated at the sixth N-glycosylation site. For example, the sixth N-glycosylation site has a mixture of complex two-, three-, or four-headed fucosylated glycans as the major species. In some embodiments, at least 3%, 5%, 8%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the rhGAA is sialylated at the sixth N-glycosylation site.

[0117] In certain embodiments, at least 5% of the rhGAA is phosphorylated at the seventh N-glycosylation site (e.g., N869 for SEQ ID NO: 2 and N925 for SEQ ID NO: 1). In other embodiments, less than 5%, 10%, 15%, 20%, or 25% of the rhGAA is phosphorylated at the seventh N-glycosylation site. In some embodiments, less than 40%, 45%, 50%, 55%, 60%, or 65% of the rhGAA has any glycan at the seventh N-glycosylation site. In some embodiments, less than 30%, 35%, or 40% of the rhGAA has a glycan at the seventh N-glycosylation site.

[0118] In various embodiments, the rhGAA has an average fucose content of 0-5 mol per mol of rhGAA, GlcNAc content of 10-30 mol per mol of rhGAA, galactose content of 5-20 mol per mol of rhGAA, mannose content of 10-40 mol per mol of rhGAA, M6P content of 2-8 mol per mol of rhGAA, and sialic acid content of 2-8 mol per mol of rhGAA. In various embodiments, rhGAA has an average fucose content of 2-3 mol per mol of rhGAA, GlcNAc content of 20-25 mol per mol of rhGAA, galactose content of 8-12 mol per mol of rhGAA, mannose content of 22-27 mol per mol of rhGAA, M6P content of 3-5 mol per mol of rhGAA, and sialic acid content of 4-7 mol of rhGAA.

[0119] The recombinant human lysosomal protein (e.g., rhGAA) is preferably produced by Chinese hamster ovary (CHO) cells, such as the CHO cell line GA-ATB-200 or ATB-200-001-X5-14, or by a subculture or derivative of such a CHO cell culture. DNA constructs, which express allelic variants of acid α-glucosidase or other variant acid α-glucosidase amino acid sequences, such as those that are at least 90%, 95%, 98%, or 99% identical to SEQ ID NO: 1 or SEQ ID NO: 2, can be constructed and expressed in CHO cells. These variant acid α-glucosidase amino acid sequences may contain deletions, substitutions, and/or insertions relative to SEQ ID NO: 1 or SEQ ID NO: 2, such as having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more deletions, substitutions, and/or insertions relative to the amino acid sequence described by SEQ ID NO: 1 or SEQ ID NO: 2. Those skilled in the art can select suitable alternative vectors for transformation of CHO cells for production of such DNA constructs.

[0120] Various alignment algorithms and/or programs may be used to calculate the identity between two sequences, including FASTA or BLAST, which are available as a part of the GCG sequence analysis package (University of Wisconsin, Madison, Wis.) and may be used with, e.g., standard definition. For example, polypeptides having at least 90%, 95%, 98%, or 99% identity to specific polypeptides described herein and preferably exhibiting substantially the same functions as the polynucleotide encoding such polypeptides are contemplated. Unless otherwise indicated, a similarity score will be based on the use of BLOSUM62. When BLASTP is used, the percentage similarity is based on the BLASTP positive score and the percentage sequence identity is based on the BLASTP identity score. BLASTP “identities” show the number and fraction of total residues in the high-scoring sequence pairs that are identical; and “Positives” BLASTP scores show the number and fraction of residues for which the alignment scores have positive values and that are similar to each other. Amino acid sequences having these degrees of identity or similarity or any intermediate degree of identity of similarity with the amino acid sequences disclosed herein are contemplated and encompassed by this disclosure. The polynucleotide sequences of similar polypeptides are deduced using the genetic code and can be obtained by conventional means, in particular by reverse translation of their amino acid sequence using the genetic code.

[0121] The inventors have discovered that recombinant human acid α-glucosidase having superior ability to target cation-independent mannose-6-phosphate receptors (CIMPR) and cellular lysosomes as well as glycosylation patterns that reduce its nonproductive clearance in vivo can be produced using Chinese hamster ovary (CHO) cells. These cells can be induced to express recombinant human acid α-glucosidase with significantly higher levels of N-glycan units carrying one or more mannose-6-phosphate residues than conventional recombinant human acid α-glucosidase products such as alglucosidase alfa. Recombinant human acid α-glucosidase produced by these cells, for example, as exemplified by ATB200, has significantly more mannose-6-phosphate (M6P) and bis-mannose-6-phosphate N-glycan residues targeting muscle cells than conventional acid α-glucosidase such as Lumizyme®. Without being bound by theory it is believed that this extensive glycosylation allows the ATB200 enzyme to be taken up more efficiently into target cells and therefore to be cleared from the circulation more efficiently than other recombinant human acid α-glucosidases, such as, for example, alglucosidase alfa, which has a much lower M6P and bis-M6P content. ATB200 has been shown to bind effectively to CIMPR and is efficiently taken up by skeletal muscle and cardiac muscle and has a glycosylation pattern that provides a favorable pharmacokinetic profile and reduces nonproductive clearance in vivo.

[0122] It is also contemplated that the extensive glycosylation of ATB200 may contribute to a reduction in the immunogenicity of ATB200 compared to, for example, alglucosidase alfa. As will be appreciated by those skilled in the art, glycosylation of proteins with conserved mammalian sugars generally enhances product solubility and decreases product aggregation and immunogenicity. Glycosylation indirectly alters the immunogenicity of the protein by minimizing protein aggregation as well as by shielding immunogenic protein epitopes from the immune system (Guidance for Industry - Immunogenicity Assessment for Therapeutic Protein Products, US Department of Health and Human Services, Food and Drug Administration, Center for Drug Evaluation and Research, Center for Biologics Evaluation and Research, August 2014). Therefore, in at least one embodiment, administration of recombinant human acid α-glucosidase does not induce anti-drug antibodies. In at least one embodiment, administration of recombinant human acid α-glucosidase induces a lower incidence of anti-drug antibodies in a subject than the level of anti-drug antibodies induced by administration of alglucosidase alfa.

[0123] As described in copending international patent application PCT/US2015/053252, cells such as CHO cells can be used to produce the rhGAA described herein and such rhGAA can be used in the present invention. Examples of such a CHO cell line are GA-ATB-200 or ATB-200-001-X5-14 or a subculture thereof that produces a rhGAA composition as described therein. Such CHO cell lines can contain multiple copies of a gene, such as 5, 10, 15 or 20 or more copies, of a polynucleotide encoding GAA.

[0124] M6P- and bis-M6P-rich rhGAA, such as rhGAA ATB200, can be produced by transforming CHO cells with a DNA construct encoding GAA. Although CHO cells have previously been used to produce rhGAA, it has not been found that transformed CHO cells could be cultured and selected in a manner that would produce rhGAA having a high content of M6P and bis-M6P glycans that target CIMPR.

[0125] Surprisingly it was found that it was possible to transform CHO cell lines, select transformants that produce rhGAA containing a high content of M6P or bis-M6P glycans that target CIMPR and stably express this M6P-rich rhGAA. Accordingly, methods for manufacturing such CHO cell lines are also described in co-pending international patent application PCT/US2015/053252. This method involves transforming a CHO cell with DNA encoding GAA or a GAA variant, selecting a CHO cell that stably integrates the DNA encoding GAA into its chromosome(s) and that stably expresses GAA and selecting a CHO cell that expresses GAA having a high content of glycans carrying M6P or bis-M6P and, optionally, selecting a CHO cell having N-glycans with a high sialic acid content and/or having N-glycans with a low unphosphorylated high-mannose content.

[0126] These CHO cell lines can be used to produce rhGAA and rhGAA compositions by culturing the CHO cell line and recovering said composition from the CHO cell culture. Production, Capture and Purification of Recombinant Human Lysosomal Protein

[0127] Various embodiments of the present invention relate to methods for producing and/or capturing and/or purifying recombinant human lysosomal protein (e.g., rhGAA). An exemplary process 600 for producing, capturing, and purifying recombinant human lysosomal protein is shown in Figure 6.

[0128] In Figure 6, arrows indicate the direction of movement for various liquid phases containing recombinant human lysosomal protein. Bioreactor 601 contains a culture of cells, such as CHO cells, that express and secrete recombinant human lysosomal protein (e.g., rhGAA) into the surrounding liquid culture media. Bioreactor 601 may be any bioreactor suitable for culturing the cells, such as a perfusion, batch, or fed-batch bioreactor. In various embodiments, the bioreactor has a volume between about 1 L and about 20,000 L. Exemplary bioreactor volumes include about 1 L, about 10 L, about 20 L, about 30 L, about 40 L, about 50 L, about 60 L, about 70 L, about 80 L, about 90 L, about 100 L, about 150 L, about 200 L, about 250 L, about 300 L, about 350 L, about 400 L, about 500 L, about 600 L, about 700 L, about 800 L, about 900 L, about 1,000 L, about 1,500 L, about 2,000 L, about 3,500 L, or about 4,000 L. 2,500 L, about 3,000 L, about 3,500 L, about 4,000 L, about 5,000 L, about 6,000 L, about 7,000 L, about 8,000 L, about 9,000 L, about 10,000 L, about 15,000 L and about 20,000 L.

[0129] As shown in Figure 6 , media may be removed from the bioreactor. Such media removal may be continuous for a perfusion bioreactor or may be batch-by-batch for a fed-batch or batch reactor. The media is filtered by filtration system 603 to remove cells. In some embodiments, cells removed from the media are reintroduced into the bioreactor and the media comprising the secreted recombinant human lysosomal protein may be further processed. Filtration system 603 may be any filtration system, including an alternating tangential flow filtration (ATF) system, a tangential flow filtration (TFF) system, centrifugal filtration system, etc. In various embodiments, the filtration system utilizes a filter having a pore size between about 10 nanometers and about 2 micrometers. Exemplary filter pore sizes include about 10 nm, about 20 nm, about 30 nm, about 40 nm, about 50 nm, about 60 nm, about 70 nm, about 80 nm, about 90 nm, about 100 nm, about 150 nm, about 200 nm, about 250 nm, about 300 nm, about 350nm, about 400nm, about 500nm, about 600nm, about 700nm, about 800nm, about 900nm, about 1μm, about 1.5μm and about 2μm.

[0130] In various embodiments, the media removal rate is between about 1 L/day and about 20,000 L/day. Exemplary media removal rates include about 1 L/day, about 10 L/day, about 20 L/day, about 30 L/day, about 1,000 L/day, about 1,500 L/day, about 2,000 L/day, about 2,500 L/day, about 3,000 L/day, about 3,500 L/day, about 4,000 L/day, about 5,000 L/day, about 6,000 L/day, about 7,000 L/day, about 8,000 L/day, about 9,000 L/day, about 10,000 L/day, about 15,000 L/day, and about 20,000 L/day. Alternatively, the media removal rate can be expressed as a function of bioreactor volume, such as about 0.1 to about 3 reactor volumes per day. Exemplary media removal rates include about 0.1, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1, about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 2, about 2.5, and about 3 reactor volumes per day.

[0131] For a continuous or fed-batch process, the rate at which fresh media is provided to the bioreactor can be between about 1 L/day and about 20,000 L/day. Exemplary media introduction rates include about 1 L/day, about 10 L/day, about 20 L/day, about 30 L/day, about 40 L/day, about 50 L/day, about 60 L/day, about 70 L/day, about 80 L/day, about 90 L/day, about 100 L/day, about 150 L/day, about 200 L/day, about 250 L/day, about 300 L/day, about 350 L/day, about 400 L/day, about 500 L/day, about 600 L/day, about 700 L/day, about 800 L/day, about 900 L/day, about 1,000 L/day, about 1,500 L/day, and so on. L/day, about 2,000 L/day, about 2,500 L/day, about 3,000 L/day, about 3,500 L/day, about 4,000 L/day, about 5,000 L/day, about 6,000 L/day, about 7,000 L/day, about 8,000 L/day, about 9,000 L/day, about 10,000 L/day, about 15,000 L/day, and about 20,000 L/day. Alternatively, the media introduction rate can be expressed as a function of bioreactor volume, such as about 0.1 to about 3 reactor volumes per day. Exemplary media introduction rates include about 0.1, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1, about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 2, about 2.5, and about 3 reactor volumes per day.

[0132] After filtration, the filtrate is loaded into a protein capture system 605. The protein capture system 605 may include one or more chromatography columns. If more than one chromatography column is used, then the columns may be placed in series such that the next column can begin loading as soon as the first column is loaded. Alternatively, the media removal process may be stopped while the columns are changed.

[0133] In various embodiments, the protein capture system 605 includes one or more anion exchange (AEX) columns for the direct capture of recombinant human lysosomal protein product, particularly lysosomal protein having a high M6P content. While not being limited by any particular theory, it is believed that the use of AEX chromatography to capture recombinant human lysosomal protein from filtered media ensures that the captured recombinant protein product has a higher M6P content, due to the more negative charge of the recombinant protein having one or more M6P groups. As a result, the non-phosphorylated recombinant protein and host cell impurities do not bind to the column resin as well as the highly phosphorylated recombinant protein, and the non-phosphorylated recombinant protein and host cell impurities pass through the column. Accordingly, AEX chromatography can be used to enrich the M6P content of the protein product (i.e., select proteins having more M6P) due to the high affinity of M6P-containing proteins for the AEX resin.

[0134] Furthermore, while not being limited by any particular theory, it is also believed that having a direct product capture of recombinant protein using AEX chromatography ensures that recombinant proteins having high M6P content are removed from media containing proteases and other enzymes that can degrade the protein and/or dephosphorylate the protein. As a result, high quality product is preserved.

[0135] AEX chromatography columns have chemical functional groups that negatively bind to charged proteins. Exemplary functional groups include, but are not limited to, primary, secondary, tertiary, and quaternary ammonium or amine groups. These functional groups can be attached to membranes (e.g., cellulose membranes) or conventional chromatography resins. Exemplary culture media include SP, CM, Q, and DEAE Sepharose® Fast Flow media from GE Healthcare Lifesciences.

[0136] The volume of the AEX chromatography column can be any suitable volume, such as between 1 L and 1,000 L. Exemplary column volumes include about 1 L, about 2 L, about 3 L, about 4 L, about 5 L, about 6 L, about 7 L, about 8 L, about 9 L, about 10 L, about 20 L, about 30 L, about 40 L, about 50 L, about 60 L, about 70 L, about 80 L, about 90 L, about 100 L, about 150 L, about 200 L, about 250 L, about 300 L, about 350 L, about 400 L, and about 500 L, about 600 L, about 700 L, or about 800 L. L, about 800 L, about 900 L and about 1,000 L.

[0137] Exemplary conditions for a cation exchange column are provided in Table 2 below:

[0138] After the recombinant human lysosomal protein is loaded into the protein capture system 605, the recombinant human lysosomal protein is eluted from the column(s) by changing the pH and/or salt content in the column.

[0139] The eluted recombinant human lysosomal protein may be subjected to additional purification steps and/or quality assurance steps. For example, as shown in Figure 6, the eluted recombinant human lysosomal protein may be subjected to a virus killing step 607. Such a virus killing 607 may include one or more of a low pH killing, a detergent killing, or another technique known in the art.

[0140] The recombinant protein product from virus killing step 607 may be introduced into a second chromatography system 609 to further purify the recombinant protein product. Alternatively, recombinant protein eluted from protein capture system 605 may be fed directly to second chromatography system 609. In various embodiments, second chromatography system 609 includes one or more immobilized metal affinity chromatography (IMAC) columns for further removal of impurities. Exemplary metal ions include cobalt, nickel, copper, iron, zinc, or gallium.

[0141] The volume of the second chromatography column (e.g., IMAC column) can be any suitable volume, such as between 0.1 L and 100 L. Exemplary column volumes include about 0.1 L, about 0.2 L, about 0.3 L, about 0.4 L, about 0.5 L, about 0.6 L, about 0.7 L, about 0.8 L, about 0.9 L, about 1 L, about 1.5 L, about 2 L, about 2.5 L, about 3 L, about 3.5 L, about 4 L, about 4.5 L, about 5 L, about 6 L, about 7 L, about 8 L, about 9 L, about 10 L, about 15 L, about 20 L, about 25 L, about from 30 L, about 35 L, about 40 L and about 50 L, about 60 L, about 70 L, about 80 L, about 90 L and about 100 L.

[0142] Exemplary conditions for an IMAC column are provided in Table 3 below: Table 3

[0143] After the recombinant protein is loaded onto the second chromatography system 609, the recombinant protein is eluted from the column(s). As shown in Figure 6 , the eluted recombinant protein may be subjected to a virus killing step 611. As with virus killing 607, virus killing 611 may include one or more of a low pH kill, a detergent kill, or another technique known in the art. In some embodiments, only one of virus killing 607 or 611 is used, or the virus kills are performed at the same step in the purification process.

[0144] As shown in Figure 6 , the recombinant protein product from virus killing step 611 may be introduced into a third chromatography system 613 to further purify the recombinant protein product. Alternatively, recombinant protein eluted from second chromatography system 609 may be fed directly to third chromatography system 613. In various embodiments, third chromatography system 613 includes one or more cation exchange chromatography (CEX) columns and/or size exclusion chromatography (SEC) columns for further removal of impurities. The recombinant protein product is then eluted from third chromatography system 613.

[0145] The volume of the third chromatography column (e.g., CEX or SEC column) can be any suitable volume, such as between 0.1 L and 200 L. Exemplary column volumes include about 0.1 L, about 0.2 L, about 0.3 L, about 0.4 L, about 0.5 L, about 0.6 L, about 0.7 L, about 0.8 L, about 0.9 L, about 1 L, about 1.5 L, about 2 L, about 2.5 L, about 3 L, about 3.5 L, about 4 L, about 4.5 L, about 5 L, about 6 L, about 7 L, about 8 L, about 9 L, about 10 L, about 15 L, about 20 L, about 25 L, or about 30 L. L, about 30 L, about 35 L, about 40 L and about 50 L, about 60 L, about 70 L, about 80 L, about 90 L, about 100 L, about 150 L and about 200 L.

[0146] Exemplary conditions for a CEX column are provided in Table 4 below: Table 4

[0147] The recombinant protein product may also be subjected to further processing. For example, another filtration system 615 may be used to remove viruses. In some embodiments, such filtration may utilize filters with pore sizes between 5 and 50 μm. Further product processing may include a product adjustment step 617, in which the recombinant protein product may be sterilized, filtered, concentrated, stored, and/or have additional components added to the final product formulation. For example, the recombinant protein product may be concentrated by a factor of 2-10-fold, such as from a starting protein concentration of about 2 to about 20 mg/mL to a final protein concentration of about 4 to about 200 mg/mL. This final product may be used to fill vials and may be lyophilized for future use.

Recombinant Protein Administration

[0148] The recombinant human lysosomal protein (e.g., rhGAA), or a pharmaceutically acceptable salt thereof, may be formulated in accordance with routine procedures as a pharmaceutical composition adapted for administration to humans. For example, in a preferred embodiment, a composition for intravenous administration is a solution in sterile isotonic aqueous buffer. Where necessary, the composition may also include a solubilizing agent and a local anesthetic to alleviate pain at the injection site. Generally, the ingredients are supplied separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water-free concentrate in a hermetically sealed container such as an ampoule or sachet indicating the amount of active agent. Where the composition is to be administered by infusion it may be dispensed with an infusion bottle containing sterile pharmaceutical grade water, saline or dextrose/water. Where the composition is administered by injection an ampoule of sterile water for injection or saline solution may be provided such that the ingredients can be mixed prior to administration.

[0149] The recombinant human lysosomal protein (e.g., rhGAA) (or a composition or medicament containing recombinant human lysosomal protein) is administered by an appropriate route. In one embodiment, the recombinant human lysosomal protein is administered intravenously. In other embodiments, the recombinant human lysosomal protein (e.g., rhGAA) is administered by direct administration to a target tissue, such as cardiac or skeletal muscle (e.g., intramuscularly) or nervous system (e.g., direct injection into the brain, intraventricularly, intrathecally). More than one route may be used simultaneously, if desired.

[0150] The recombinant human lysosomal protein (e.g., rhGAA) (or a composition or medicament containing recombinant human lysosomal protein) is administered in a therapeutically effective amount (e.g., a dosage amount that, when administered at regular intervals, is sufficient to treat the disease, such as by ameliorating symptoms associated with the disease, preventing or delaying the onset of the disease, and/or attenuating the severity or frequency of symptoms of the disease). The amount that will be therapeutically effective in treating the disease will depend on the nature and extent of the effects of the disease and can be determined by standard clinical techniques. Additionally, in vitro or in vivo assays may optionally be employed to help identify optimal dosage ranges. The precise dose to be employed will also depend on the route of administration and the severity of the disease and should be decided in accordance with a physician's judgment and the circumstances of the individual patient. Effective doses can be extrapolated from dose-response curves derived from in vitro or animal model test systems. In at least one embodiment, recombinant human acid α-glucosidase is administered by intravenous infusion at a dose of about 1 mg/kg to about 100 mg/kg, such as about 5 mg/kg to about 30 mg/kg, typically about 5 mg/kg to about 20 mg/kg. In at least one embodiment, recombinant human acid α-glucosidase is administered by intravenous infusion at a dose of about 5 mg/kg, about 10 mg/kg, about 15 mg/kg, about 20 mg/kg, about 25 mg/kg, about 30 mg/kg, about 35 mg/kg, about 40 mg/kg, about 50 mg/kg, about 50 mg/kg, about 60 mg/kg, about 70 mg/kg, about 80 mg/kg, about 90 mg/kg, or about 100 mg/kg. In at least one embodiment, recombinant human acid α-glucosidase is administered by intravenous infusion at a dose of about 20 mg/kg. The effective dose for a particular individual can be varied (e.g., increased or decreased) over time, depending on the needs of the individual. For example, in periods of physical illness or stress, or if anti-acid α-glucosidase antibodies become present or increase, or if symptoms of the disease worsen, the amount may be increased.

[0151] The therapeutically effective amount of recombinant human acid α-glucosidase (or composition or drug containing recombinant human acid α-glucosidase) is administered at regular intervals, depending on the nature and extent of the effects of the disease, and on a continuous basis. Administration at a “regular interval,” as used herein, indicates that the therapeutically effective amount is administered periodically (as distinct from a single dose). The interval can be determined by standard clinical techniques. In preferred embodiments, recombinant human acid α-glucosidase is administered monthly, bimonthly; weekly; twice weekly; or daily. The interval of administration for a single individual need not be a fixed interval, but may be varied over time, depending on the needs of the individual. For example, in periods of physical illness or stress, if anti-human recombinant acid α-glucosidase antibodies become present or increase, or if symptoms of the disease worsen, the dosing interval may be decreased. In some embodiments, a therapeutically effective amount of 5, 10, 20, 50, 100, or 200 mg enzyme/kg body weight is administered twice weekly, weekly, or every other week with or without a chaperone.

[0152] The recombinant human lysosomal protein (e.g., rhGAA) may be prepared for later use, such as in a unit dose vial or syringe, or in a bottle or bag for intravenous administration. Kits containing the recombinant human lysosomal protein (e.g., rhGAA), as well as optional excipients or other active ingredients, such as chaperones or other drugs, may be enclosed in packaging material and accompanied by instructions for reconstitution, dilution, or dosing for treatment of a subject in need of treatment, such as a patient having Pompe disease.

Combination Therapy of rhGAA and Pharmacological Chaperone

[0153] In various embodiments, rhGAA (e.g., ATB200) produced by the processes described herein can be used in combination therapy with a pharmacological chaperone such as miglustat or duvoglustat.

[0154] In at least one embodiment, the pharmacological chaperone (e.g., miglustat) is administered orally. In at least one embodiment, miglustat is administered at an oral dose of about 200 mg to about 400 mg, or at an oral dose of about 200 mg, about 250 mg, about 300 mg, about 350 mg, or about 400 mg. In at least one embodiment, miglustat is administered at an oral dose of about 233 mg to about 400 mg. In at least one embodiment, miglustat is administered at an oral dose of about 250 mg to about 270 mg, or at an oral dose of about 250 mg, about 255 mg, about 260 mg, about 265 mg, or about 270 mg. In at least one embodiment, miglustat is administered as an oral dose of approximately 260 mg.

[0155] It will be understood by those skilled in the art that an oral dose of miglustat in the range of about 200 mg to 400 mg or any smaller range thereof may be suitable for an adult patient with an average body weight of about 70 kg. For patients having a body weight significantly lower than about 70 kg, including but not limited to infants, children or underweight adults, a smaller dose may be considered suitable by a physician. Therefore, in at least one embodiment, miglustat is administered as an oral dose of about 50 mg to about 200 mg, or as an oral dose of about 50 mg, about 75 mg, about 100 mg, 125 mg, about 150 mg, about 175 mg or about 200 mg. In at least one embodiment, miglustat is administered as an oral dose of about 65 mg to about 195 mg, or as an oral dose of about 65 mg, about 130 mg, or about 195 mg.

[0156] In at least one embodiment, miglustat is administered as a pharmaceutically acceptable dosage form suitable for oral administration and includes, but is not limited to, tablets, capsules, ovules, elixirs, solutions or suspensions, gels, syrups, mouthwashes, or a dry powder for reconstitution with water or other suitable vehicle before use, optionally with flavoring and coloring agents for immediate, delayed, modified, sustained, pulsed, or controlled release applications. Solid compositions such as tablets, capsules, lozenges, pills, boluses, powders, pastes, granules, candies, dragees, or premixed preparations may also be used. In at least one embodiment, miglustat is administered as a tablet. In at least one embodiment, miglustat is administered as a capsule. In at least one embodiment, the dosage form contains from about 50 mg to about 300 mg of miglustat. In at least one embodiment, the dosage form contains about 65 mg of miglustat. In at least one embodiment, the dosage form contains about 130 mg of miglustat. In at least one embodiment, the dosage form contains about 260 mg of miglustat. It is contemplated that when the dosage form contains about 65 mg of miglustat, miglustat may be administered as a dosage of four dosage forms or a total dose of 260 mg of miglustat. However, for patients who have a weight significantly lower than an average adult weight of 70 kg, including but not limited to infants, children, or underweight adults, miglustat may be administered as a dosage of one dosage form (a total dose of 65 mg miglustat), two dosage forms (a total dose of 130 mg miglustat), or three dosage forms (a total dose of 195 mg miglustat).

[0157] Solid and liquid compositions for oral use may be prepared according to methods well known in the art. Such compositions may also contain one or more pharmaceutically acceptable carriers and excipients which may be in solid or liquid form. Tablets or capsules may be prepared by conventional means with pharmaceutically acceptable excipients, including but not limited to binding agents, fillers, lubricants, disintegrants or wetting agents. Suitable pharmaceutically acceptable excipients are known in the art and include, but are not limited to, pregelatinized starch, polyvinylpyrrolidone, povidone, hydroxypropyl methyl cellulose (HPMC), hydroxypropyl ethyl cellulose (HPEC), hydroxypropyl cellulose (HPC), sucrose, gelatin, acacia, lactose, microcrystalline cellulose, calcium hydrogen phosphate, magnesium stearate, stearic acid, glyceryl behenate, talc, silica, corn, potato or tapioca starch, sodium starch glucolate, sodium lauryl sulfate, sodium citrate, calcium carbonate, dibasic calcium phosphate, croscarmellose sodium glycine, and complex silicates. Tablets may be coated by methods well known in the art. In at least one embodiment, miglustat is administered as a commercially available formulation as Zavesca® (Actelion Pharmaceuticals).

[0158] In at least one embodiment, miglustat and recombinant human acid α-glucosidase are administered simultaneously. In at least one embodiment, miglustat and recombinant human acid α-glucosidase are administered sequentially. In at least one embodiment, miglustat is administered prior to administration of recombinant human acid α-glucosidase. In at least one embodiment, miglustat is administered less than three hours prior to administration of recombinant human acid α-glucosidase. In at least one embodiment, miglustat is administered about two hours prior to administration of recombinant human acid α-glucosidase. In at least one embodiment, miglustat is administered less than two hours prior to administration of recombinant human acid α-glucosidase. In at least one embodiment, miglustat is administered about 1.5 hours prior to administration of recombinant human acid α-glucosidase. In at least one embodiment, miglustat is administered about one hour prior to administration of recombinant human acid α-glucosidase. In at least one embodiment, miglustat is administered about 50 minutes to about 70 minutes prior to administration of recombinant human acid α-glucosidase. In at least one embodiment, miglustat is administered about 55 minutes to about 65 minutes prior to administration of recombinant human acid α-glucosidase. In at least one embodiment, miglustat is administered about 30 minutes prior to administration of recombinant human acid α-glucosidase. In at least one embodiment, miglustat is administered from about 25 minutes to about 35 minutes prior to administration of recombinant human acid α-glucosidase. In at least one embodiment, miglustat is administered from about 27 minutes to about 33 minutes prior to administration of recombinant human acid α-glucosidase.

[0159] In at least one embodiment, miglustat is administered simultaneously with the administration of recombinant human acid α-glucosidase. In at least one embodiment, miglustat is administered within 20 minutes prior to or following the administration of recombinant human acid α-glucosidase. In at least one embodiment, miglustat is administered within 15 minutes prior to or following the administration of recombinant human acid α-glucosidase. In at least one embodiment, miglustat is administered within 10 minutes prior to or following the administration of recombinant human acid α-glucosidase. In at least one embodiment, miglustat is administered within 5 minutes prior to or following the administration of recombinant human acid α-glucosidase.

[0160] In at least one embodiment, miglustat is administered after administration of recombinant human acid α-glucosidase. In at least one embodiment, miglustat is administered up to 2 hours after administration of recombinant human acid α-glucosidase. In at least one embodiment, miglustat is administered about 30 minutes after administration of recombinant human acid α-glucosidase. In at least one embodiment, miglustat is administered about 1 hour after administration of recombinant human acid α-glucosidase. In at least one embodiment, miglustat is administered about 1.5 hours after administration of recombinant human acid α-glucosidase. In at least one embodiment, miglustat is administered about 2 hours after administration of recombinant human acid α-glucosidase.

[0161] Another aspect of the invention provides a kit for combination therapy of Pompe disease in a patient in need thereof. The kit includes a pharmaceutically acceptable dosage form comprising miglustat, a pharmaceutically acceptable dosage form comprising a recombinant human acid α-glucosidase as defined herein, and instructions for administering the pharmaceutically acceptable dosage form comprising miglustat and the pharmaceutically acceptable dosage form comprising recombinant acid α-glucosidase to a patient in need thereof. In at least one embodiment, the pharmaceutically acceptable dosage form comprising miglustat is an oral dosage form as described herein, including but not limited to a tablet or a capsule. In at least one embodiment, the pharmaceutically acceptable dosage form comprising a recombinant human acid α-glucosidase is a sterile solution suitable for injection as described herein. In at least one embodiment, the instructions for administering the dosage forms include instructions for administering the pharmaceutically acceptable dosage form comprising miglustat orally prior to administering the pharmaceutically acceptable dosage form comprising recombinant human acid α-glucosidase by intravenous infusion, as described herein.

[0162] Without being limited by theory, it is believed that miglustat acts as a pharmacological chaperone for recombinant human α-glucosidase ATB200 and binds to its active site. For example, miglustat has been found to decrease the percentage of unfolded ATB200 protein and stabilize the active conformation of ATB200, preventing irreversible denaturation and inactivation at neutral plasma pH and allowing it to survive circulating conditions long enough to reach and be absorbed by tissues. However, binding of miglustat to the active site of ATB200 may also result in inhibition of the enzymatic activity of ATB200 by preventing access of the natural substrate, glycogen, to the active site. It is believed that when miglustat and recombinant human acid α-glucosidase are administered to a patient under the conditions described herein, the concentrations of miglustat and ATB200 in plasma and tissues are such that ATB200 is stabilized until it can be taken up by tissues and targeted to lysosomes, but because of the rapid clearance of miglustat, the hydrolysis of glycogen by ATB200 within lysosomes is not unduly inhibited by the presence of miglustat, and the enzyme retains sufficient activity to be therapeutically useful.

[0163] All of the above described embodiments may be combined. This includes in particular embodiments relating to: • the nature of the pharmacological chaperone, e.g. miglustat; and the active site for which it is specific; • the dosage, route of administration of the pharmacological chaperone (e.g. miglustat) and the type of pharmaceutical composition including the nature of the carrier and the use of commercially available compositions; • the nature of the drug, e.g., therapeutic protein drug product, which may be a counterpart of an endogenous protein for which expression is reduced or absent in the subject, suitably recombinant human lysosomal protein (e.g., rhGAA), for example recombinant human lysosomal protein (e.g., rhGAA) expressed in Chinese hamster ovary (CHO) cells and comprising an increased content of N-glycan units carrying one or more mannose-6-phosphate residues as compared to a content of N-glycan units carrying one or more mannose-6-phosphate residues of alglucosidase alfa; and suitably having an amino acid sequence as set forth in SEQ ID NO: 1 or SEQ ID NO: 2; • the number and type of N-glycan units on the recombinant human lysosomal protein (e.g., rhGAA), such as N-acetylglucosamine, galactose, sialic acid, or complex N-glycans formed from combinations of these, attached to the recombinant human lysosomal protein; • the degree of phosphorylation of mannose units on the recombinant human lysosomal protein (e.g., rhGAA) to form mannose-6-phosphate and/or bis-mannose-6-phosphate; • the dosage and route of administration (e.g., intravenous administration, especially intravenous infusion, or direct administration to the target tissue) of the replacement enzyme (e.g., recombinant human acid α-glucosidase) and the type of formulation including carriers and therapeutically effective amount; • the dosage range of the pharmacological chaperone (miglustat) and recombinant human acid α-glucosidase; • the nature of the therapeutic response and the results of the combination therapy (e.g. enhanced results compared with the effect of each therapy given individually); • the timing of administration of the combination therapy, e.g. simultaneous administration of miglustat and recombinant human acid α-glucosidase or sequential administration, for example where miglustat is administered before recombinant human acid α-glucosidase or after recombinant human acid α-glucosidase or within a certain period of time before or after administration of recombinant human acid α-glucosidase; and • the nature of the patient treated (e.g. mammal such as human) and the condition suffered by the individual (e.g. enzyme insufficiency).

[0164] Any of the modalities in the above list may be combined with one or more of the other modalities in the list.

EXAMPLES

[0165] Other features of the present invention will become apparent from the following non-limiting examples which illustrate, by way of example, the principles of the invention.

Example 1: Limitations of existing rhGAA products Myozyme® and Lumizyme®

[0166] To evaluate the capacity of rhGAA in Myozyme® and Lumizyme®, the only currently approved treatments for Pompe disease, these rhGAA preparations were injected onto a CIMPR column (which binds rhGAA having M6P groups) and subsequently eluted with an M6-free gradient. Fractions were collected in a 96-well plate and GAA activity was assayed by 4MU-α-glucose substrate. The relative amounts of bound and unbound rhGAA were determined based on GAA activity and reported as the total enzyme fraction.

[0167] Figures 4A-B depict the problems associated with conventional ERTs (Myozyme® and Lumizyme®): 73% of rhGAA in Myozyme® (Figure 4B) and 78% of rhGAA in Lumizyme® (Figure 4A) did not bind to CIMPR, see leftmost peaks in each figure. Only 27% of rhGAA in Myozyme® and 22% of rhGAA in Lumizyme® contained M6P that could be productive for targeting to CIMPR in muscle cells.

[0168] An effective dose of Myozyme® and Lumizyme® corresponds to the amount of M6P-containing rhGAA that targets the CIMPR on muscle cells. However, the majority of rhGAA in these two conventional products does not target the CIMPR receptor on target muscle cells. Administration of a conventional rhGAA where the majority of rhGAA is not targeted to muscle cells increases the risk of allergic reaction or induction of immunity against the non-targeted rhGAA.

Example 2: Preparation of CHO Cells Producing rhGAA ATB200 Having a High Content of N-Glycans Carrying Mono- or Bis-M6P

[0169] CHO cells were transfected with DNA expressing rhGAA followed by selection of transformants producing rhGAA. A DNA construct for transformation of CHO cells with DNA encoding rhGAA is shown in Figure 5. CHO cells were transfected with DNA expressing rhGAA followed by selection of transformants producing rhGAA.

[0170] After transfection, DG44 CHO (DHFR-) cells containing a stably integrated GAA gene were selected with hypoxanthine/thymidine (-HT) deficient medium. Amplification of the

[0171] GAA expression in these cells was induced by methotrexate (MTX, 500 nM) treatment. Cell clusters expressing high amounts of GAA were identified by GAA enzymatic activity assays and were used to establish individual clones producing rhGAA. Individual clones were generated on semi-solid media plates, harvested by ClonePix system and transferred to 24 deep-well plates. Individual clones were assayed for GAA enzymatic activity to identify clones expressing a high level of GAA. Conditioned media for determination of GAA activity used a 4-MU-α-Glucosidase substrate. Clones producing higher levels of GAA as measured by GAA enzymatic assays were further evaluated for viability, growth capacity, GAA productivity, N-glycan structure and stable protein expression. CHO cell lines, including the CHO cell line GA-ATB-200, expressing rhGAA with N-glycans enhanced on mono-M6P or bis-M6P were isolated using this procedure.

Example 3: Capture and Purification of rhGAA ATB200

[0172] Multiple batches of rhGAA according to the invention were produced in shake flasks and in perfusion bioreactors using the CHO cell line GA-ATB-200 and binding to CIMPR was measured. Similar binding to the CIMPR receptor (~70%) to that shown in Figure 7B and Figure 8 was observed for rhGAA ATB200 purified from different production batches indicating that rhGAA ATB200 can be consistently produced. As shown in Figures 4A, 4B, 7A and 7B, Myozyme® and Lumizyme® rhGAAs exhibited significantly less binding to CIMPR than rhGAA ATB200.

Example 4: Analytical Comparison of ATB200 with Lumizyme®

[0173] Weak anion exchange liquid chromatography (“WAX”) was used to fractionate rhGAA ATB200 according to the terminal phosphate. Elution profiles were generated by eluting the ERT with increasing amounts of salt. The profiles were monitored by UV (A280nm). rhGAA ATB200 was obtained from CHO cells and purified. Lumizyme® was obtained from a commercial source. Lumizyme® exhibited a prominent peak to the left of its elution profile. rhGAA ATB200 exhibited four prominent peaks eluting to the right of Lumizyme® (Fig. 9). This confirms that rhGAA ATB200 was phosphorylated to a greater extent than Lumizyme® since this assessment is by terminal charge rather than affinity for CIMPR.

Example 5: Characterization of rhGAA ATB200 Oligosaccharides

[0174] Purified rhGAA ATB200 and Lumizyme® glycans were evaluated by MALDI-TOF to determine the structures of individual glycans found on each ERT (Figure 10). ATB200 samples were found to contain lower amounts of unphosphorylated high-mannose type N-glycans than Lumizyme®. The higher content of M6P glycans on ATB200 than on Lumizyme® targets rhGAA ATB200 to muscle cells more effectively. The high percentage of mono- and bis-phosphorylated structures determined by MALDI agrees with the CIMPR profiles which illustrated significantly greater binding of ATB200 to the CIMPR receptor. Analysis of N-glycans by MALDI-TOF mass spectrometry confirmed that on average each ATB200 molecule contains at least one native bis-M6P N-glycan structure. This higher bis-M6P N-glycan content in rhGAA ATB200 directly correlated with high affinity binding to CIMPR in plate binding assays for the M6P receptor (KD approximately 2-4 nM) Figure 12A.

[0175] rhGAA ATB200 was also analyzed for site-specific N-glycan profiles using two different LC-MS/MS analytical techniques. In the first analysis, the protein was denatured, reduced, alkylated and digested prior to LC-MS/MS analysis. During protein denaturation and reduction, 200 μg of protein sample, 5 μL 1 mol/L Tris-HCl (50 mM final concentration), 75 μL 8 mol/L guanidime HCl (6 M final concentration), 1 μL 0.5 mol/L EDTA (5 mM final concentration), 2 μL 1 mol/L DTT (20 mM final concentration), and Milli-Q® water were added to a 1.5 mL tube to provide a total volume of 100 μL. The sample was mixed and incubated at 56 °C for 30 min in a dry bath. During alkylation, the denatured and reduced protein sample was mixed with 5 μL 1 mol/L iodoacetamide (IAM, final concentration 50 mM), then incubated at 10–30 °C in the dark for 30 min. After alkylation, 400 μL of pre-cooled acetone was added to the sample, and the mixture was frozen at −80 °C for 4 h. The sample was then centrifuged for 5 min at 13000 rpm at 4 °C, and the supernatant was removed. 400 μL of pre-cooled acetone was added to the pellets, which were then centrifuged for 5 min at 13000 rpm at 4 °C, and the supernatant was removed. The sample was then air-dried on ice in the dark to remove the acetone residue. 40 μL of 8 M urea and 160 μL of 100 mM NH4HCO3 were added to the sample to dissolve the protein. During trypsin digestion, 50 μg of protein was then added with trypsin digestion buffer to a final volume of 100 μL, and 5 μL of 0.5 mg/mL trypsin (protein to enzyme ratio 20/1 w/w) was added. The solution was mixed well and incubated overnight (16 ± 2 h) at 37 °C. 2.5 μL 20% TFA (final concentration 0.5%) was added to quench the reaction. The sample was then analyzed using a Thermo Scientific Orbitrap Velos ProTM mass spectrometer.

[0176] In the second LC-MS/MS analysis, the ATB200 sample was prepared according to a similar denaturation, reduction, alkylation and digestion procedure, except that iodoacetic acid (IAA) was used as the alkylation reagent instead of IAM and then analyzed using the Thermo Scientific Orbitrap Fusion Lumos TribidTM Mass Spectrometer.

[0177] The results of the first and second analyses are shown in Figures 11A-11H. In Figures 11A-11H, the results of the first analysis are represented by the left bar (dark gray) and the results of the second analysis are represented by the right bar (light gray). In Figures 11B-11G, the nomenclature of symbols for representation of glycans is according to Varki, A., Cummings, R.D., Esko J.D., et al., Essentials of Glycobiology, 2nd edition (2009).

[0178] As can be seen from Figures 8A-8I, the two analyses provided similar results, although there was some variation between the results. This variation may be due to a number of factors, including the instrument used and the completeness of the N-glycan analysis. For example, if some phosphorylated glycan species were not identified and/or quantified, then the total number of phosphorylated glycans may be underrepresented, and the percentage of rhGAA carrying phosphorylated glycans at that site may be underrepresented. As another example, if some non-phosphorylated glycan species were not identified and/or quantified, then the total number of non-phosphorylated glycans may be underrepresented, and the percentage of rhGAA carrying phosphorylated glycans at that site may be overrepresented. Figure 11A shows the occupancy of ATB200 N-glycosylation sites. As can be seen from Figure 11A, the first, second, third, fourth, fifth and sixth N-glycosylation sites are mostly occupied, with both assays detecting greater than 90% and up to nearly 100% of the ATB200 enzyme having a glycan detected at each potential site. However, the seventh potential N-glycosylation site is glucosylated about half of the time.

[0179] Figure 11B shows the N-glycosylation profile of the first site, N84. As can be seen from Figure 11B, the major glycan species are bis-M6P glycans. Both the first and second analyses detected that greater than 75% of ATB200 had a bis-M6P glycan at the first site.

[0180] Figure 11C shows the N-glycosylation profile of the second site, N177. As can be seen from Figure 11C, the major glycan species are mono-M6P glycans and unphosphorylated high-mannose glycans. Both the first and second analyses detected that greater than 40% of ATB200 had a mono-M6P glycan at the second site.

[0181] Figure 11D shows the N-glycosylation profile of the third site, N334. As can be seen from Figure 11D, the major glycan species are unphosphorylated high-mannose glycans, complex two-, three-, and four-antenna glycans, and hybrid glycans. Both the first and second analyses detected that more than 20% of ATB200 had a sialic acid residue at the third site.

[0182] Figure 11E shows the N-glycosylation profile of the fourth site, N414. As can be seen from Figure 11E, the major glycan species are bis-M6P and mono-MGP glycans. Both the first and second analyses detected that greater than 40% of ATB200 had a bis-M6P glycan at the fourth site. Both the first and second analyses also detected that greater than 25% of ATB200 had a mono-M6P glycan at the fourth site.

[0183] Figure 11F shows the N-glycosylation profile of the fifth site, N596. As can be seen from Figure 11F, the major glycan species are fucosylated two-antenna complex glycans. Both the first and second analyses detected that more than 70% of ATB200 had a sialic acid residue at the fifth site.

[0184] Figure 11G shows the N-glycosylation profile of the sixth site, N826. As can be seen from Figure 11G, the major glycan species are two-, three-, and four-antenna complex glycans. Both the first and second analyses detected that more than 80% of ATB200 had a sialic acid residue at the sixth site.

[0185] An analysis of glycosylation at the seventh site, N869, showed approximately 40% glycosylation, with the most common glycans being A4S3S3GF (12%), A5S3G2F (10%), A4S2G2F (8%), and A6S3G3F (8%).

[0186] Figure 11H shows a summary of phosphorylation at each of the seven potential N-glycosylation sites. As can be seen from Figure 11H, both the first and second analyses detected high levels of phosphorylation at the first, second, and fourth sites. Both analyses detected that greater than 80% of ATB200 was mono- or di-phosphorylated at the first site, greater than 40% of ATB200 was mono-phosphorylated at the second site, and greater than 80% of ATB200 was mono- or di-phosphorylated at the fourth site.

[0187] Further analysis of ATB200 glycans was performed according to a hydrophilic interaction liquid chromatography-fluorescence detection-mass spectrometry (HILIC-FLD-MS) method.

[0188] The results of the HILIC-FLD-MS analysis are provided in Table 5 below. In Table 5, the first number in the three-digit number indicates the number of branches in the glycan, the second number indicates the number of core fucose units, and the third number indicates the number of terminal sialic acid units. Using this nomenclature, “303” represents a three-antennary glycan (the first 3) with 0 core fucoses (the 2nd 0) and 3 terminal sialic acids (the last 3), “212” represents a two-antennary glycan with 1 core fucose and 2 terminal sialic acids, “404” represents a four-antennary glycan with 0 terminal fucoses and 4 terminal sialic acids, etc. Table 5

[0189] Based on this HILIC-FLD-MS analysis it is expected that ATB200 will have an average fucose content of 2-3 mol per mol of ATB200, GlcNAc content of 20-25 mol per mol of ATB200, galactose content of 8-12 mol per mol of ATB200, mannose content of 22-27 mol per mol of ATB200, M6P content of 3-5 mol per mol of ATB200 and sialic acid content of 4-7 mol of ATB200.

Example 6: Characterization of Affinity for CIMPR of ATB200

[0190] In addition to having a higher percentage of rhGAA that can bind to CIMPR, it is important to understand the quality of this interaction. Receptor binding of rhGAA ATB200 and Lumizyme® was determined using a CIMPR plate binding assay. Briefly, CIMPR-coated plates were used to capture GAA. Varying concentrations of rhGAA were applied to the immobilized receptor and unbound rhGAA was washed away. The amount of rhGAA remaining was determined by GAA activity. As shown in Figure 12A, rhGAA ATB200 bound to CIMPR significantly better than Lumizyme®.

[0191] Figure 12B shows the relative content of bis-M6P glycans in Lumizyme®, a conventional rhGAA and ATB200 according to the invention. For Lumizyme® there are on average only 10% of the molecules have a bis-phosphorylated glycan. Contrast this with ATB200 where on average every rhGAA molecule has at least one bis-phosphorylated glycan.

Example 7: ATB200 rhGAA was more efficiently internalized by fibroblasts than Lumizyme

[0192] The relative cellular uptake of rhGAA ATB200 and Lumizyme® was compared using normal and Pompe fibroblast cell lines. Comparisons involved 5-100 nM rhGAA ATB200 according to the invention with conventional rhGAA Lumizyme® at 10-500 nM. After 16 hr incubation, external rhGAA was inactivated with TRIS base and cells were washed 3 times with PBS prior to harvesting. Internalized GAA measured by 4MU-a-Glucoside hydrolysis and was plotted against total cellular protein and results appear in Figures 13A-B.

[0193] It was also shown that rhGAA ATB200 was efficiently internalized into cells (Figure 13A and 13B), respectively, showing that rhGAA ATB200 is internalized into both normal and Pompe fibroblast cells and that it is internalized to a greater degree than conventional rhGAA Lumizyme®. rhGAA ATB200 saturates cellular receptors at approximately 20 nM, whereas approximately 250 nM of Lumizyme® is required. The uptake efficiency constant (Kuptake) extrapolated from these results is 2-3 nM for ATB200 and 56 nM for Lumizyme® as shown in Figure 13C. These results suggest that rhGAA ATB200 is a well-targeted treatment for Pompe disease.

Example 8: Glycogen reduction in Gaa knockout mice

[0194] Figures 14A to 14C show the effects of alglucosidase alfa (Lumizyme®) and ATB200 administration on glycogen clearance in Gaa knockout mice. Animals were given two IV bolus administrations (every two weeks); tissues were collected two weeks after the last dose and analyzed for acid α-glucosidase activity and glycogen content.

[0195] As seen from Figures 14A through 14C, ATB200 was found to deplete tissue glycogen in acid α-glucosidase (Gaa) knockout mice in a dose-dependent manner. The 20 mg/kg dose of ATB200 consistently removed a greater proportion of stored glycogen in Gaa knockout mice than the 5 and 10 mg/kg dose levels. However, as seen in Figures 14A through 14C, ATB200 administered at 5 mg/kg showed a similar reduction in glycogen in mouse cardiac and skeletal (quadriceps and triceps) muscles as Lumizyme® administered at 20 mg/kg, while ATB200 dosed at 10 and 20 mg/kg showed significantly better reduction in skeletal muscle glycogen levels than Lumizyme®.

[0196] Figure 15 shows the effects of alglucosidase alfa (Lumizyme®) and ATB200 administration on glycogen clearance in Gaa knockout mice, as well as the effect of co-administration of ATB200 and miglustat on glycogen clearance. Twelve-week-old GAA knockout mice were treated with Lumizyme® or ATB200, 4 injections every other week of 20 mg/kg IV; miglustat was co-administered at 10 mg/kg PO 30 min prior to rhGAA as indicated. Tissues were collected 14 days after the last enzyme dose for glycogen measurement. Figure 15 shows the relative glycogen reduction in quadriceps and triceps skeletal muscle, with ATB200 providing greater glycogen reduction than Lumizyme® and ATB200/miglustat providing an even greater glycogen reduction.

Example 9: Muscle physiology and morphology in Gaa knockout mice

[0197] Gaa knockout mice were given two IV bolus administrations of recombinant human acid α-glucosidase (alglucosidase alfa or ATB200) at 20 mg/kg every other week. Miglustat was orally administered at doses of 10 mg/kg to a subset of ATB200-treated animals 30 mins prior to ATB200 administration. Control mice were treated with vehicle only. Soleus, quadriceps, and diaphragm tissue is collected two weeks after the last dose of recombinant human acid α-glucosidase. Soleus and diaphragm tissues were analyzed for glycogen levels by periodic acid-Schiff (PAS) staining and for lysosomal proliferation by measuring levels of the marker lysosome-associated membrane protein 1 (LAMP1), which is upregulated in Pompe disease. Semithin sections of quadriceps muscle embedded in epoxy resin (Epon) were stained with methylene blue and observed by electron microscopy (1000×) to determine the extent of vacuoles. Quadriceps muscle samples were analyzed immunohistochemically to determine levels of the autophagy markers phosphatidylethanolamine conjugate of microtubule-associated protein 1A/1B light chain 3 (LC3A II) and p62, the insulin-dependent glucose transporter GLUT4, and the insulin-independent glucose transporter GLUT1.

[0198] In a similar study, Gaa knockout mice were given four IV bolus administrations of recombinant human acid α-glucosidase (alglucosidase alfa or ATB200) at 20 mg/kg every other week. Miglustat was orally administered at doses of 10 mg/kg to a subset of ATB200-treated animals 30 mins prior to ATB200 administration. Control mice were treated with vehicle alone. Cardiac muscle tissue was harvested two weeks after the last dose of recombinant human acid α-glucosidase and analyzed for glycogen levels by periodic acid-Schiff (PAS) staining and for lysosomal proliferation by measurement of LAMP1 levels.

[0199] As seen in Figure 16, administration of ATB200 showed a reduction in lysosomal proliferation in cardiac, diaphragmatic and skeletal (soleus) muscle tissue compared to conventional alglucosidase alfa treatment, and co-administration of miglustat with ATB200 showed a significant additional reduction in lysosomal proliferation, approaching levels seen in wild-type (WT) mice. Additionally, as seen in Figure 17, administration of ATB200 showed a reduction in punctate glycogen levels in cardiac and skeletal (soleus) muscle tissue compared to conventional alglucosidase alfa treatment, and co-administration of miglustat with ATB200 showed a significant additional reduction, again approaching levels seen in wild-type (WT) mice.

[0200] Likewise, as seen in Figure 18, co-administration of miglustat with ATB200 significantly reduced the number of muscle fiber vacuoles in the quadriceps of Gaa knockout mice compared to untreated mice and mice treated with alglucosidase alfa. As seen in Figure 19, both LC3II and p62 levels are increased in Gaa knockout mice compared to wild-type mice, but are significantly reduced following treatment with ATB200 and miglustat, indicating that the increase in autophagy associated with acid α-glucosidase deficiency is reduced following co-administration of ATB200 and miglustat. Additionally, levels of the insulin-dependent glucose transporter GLUT4 and the insulin-independent glucose transporter GLUT1 are increased in Gaa knockout mice compared with wild-type mice, but again are significantly reduced after treatment with ATB200 and miglustat. The elevated GLUT4 and GLUT1 levels associated with acid α-glucosidase deficiency may contribute to increased glucose uptake in muscle fibers and increased glycogen synthesis both basally and after food intake. Thus, combination treatment with ATB200 and miglustat was found to improve skeletal muscle morphology and physiology in a mouse model of Pompe disease.

Example 10: Muscle function in Gaa knockout mice

[0201] In longer-term studies of 12 biweekly administrations, ATB200 at 20 mg/kg plus miglustat at 10 mg/kg progressively increased functional muscle strength in Gaa KO mice from baseline as measured by both grip strength and wire support tests (Figures 21A-21B). Mice treated with alglucosidase alfa (Lumizyme®) receiving the same dose of ERT (20 mg/kg) were observed to decline under identical conditions throughout the majority of the study (Figures 21A-21B). As with the shorter-term study, ATB200/miglustat had substantially better glycogen clearance after 3 months (Figures 22A-22C) and 6 months (Figures 22D-22G) of treatment than alglucosidase alfa. ATB200/miglustat also reduced autophagy and intracellular accumulation of LAMP1 and dysferlin after 3 months of treatment (Figure 23) compared to alglucosidase alfa. In Figure 21A, * indicates statistical significance compared to Lumizyme® alone (p<0.05, 2-sided t-test). In Figures 22A-22G, * indicates statistical significance compared to Lumizyme® alone (p<0.05, multiple comparison using Dunnett's method under one-way ANOVA).

[0202] Taken together, these data indicate that ATB200/miglustat was effectively targeted to muscle to reverse cellular dysfunction and improve muscle function. Importantly, the apparent improvements in muscle architecture and autophagy and reduced intracellular accumulation of LAMP1 and dysferlin may be good surrogates for improved muscle physiology that correlate with improvements in functional muscle strength. These results suggest that monitoring autophagy and these key muscle proteins may be a practical, rational method to assess the efficacy of therapeutic treatments for Pompe disease in Gaa KO mice that may prove to be useful biomarkers from muscle biopsies in clinical studies.

[0203] Figure 23 shows that 6 months of ATB200 administration with or without miglustat decreased intracellular dystrophin accumulation in Gaa KO mice. There was a greater reduction in dystrophin accumulation for ATB200 ± miglustat than with Lumizyme®.

Example 11: Capture of rhα-Gal A

[0204] The CIMPR binding profile of recombinant human α-galactosidase A (rhα-Gal A) in spent cell culture medium was measured prior to product capture using AEX chromatography (Figure 20A) and after product capture using AEX chromatography (Figure 20B). The dashed lines in both graphs refer to the M6P elution gradient. Prior to product capture by AEX, 80% of rhα-Gal A is able to bind to CIMPR. After product capture by AEX, the total rhα-Gal A bound increases to 96%.

Example 12: Pharmacokinetic and Safety Data on Recombinant α-Acid Glucosidase ATB200 Coadministered With Miglustat in ERT-Experienced and ERT-Naive Patients With Pompe Disease

[0205] This study was designed to primarily evaluate the safety, tolerability, and pharmacokinetics (PK) of ATB200 co-administered with miglustat. A Gaa knockout mouse PK/pharmacodynamic (PD) translational model predicted that a combination of 20 mg/kg ATB200 with a high dose (e.g., 260 mg) of miglustat in humans would provide optimal glycogen reduction.

[0206] In the description below, “high dose” miglustat refers to a dose of about 260 mg and “low dose” miglustat refers to a dose of about 130 mg.

[0207] The objective was to evaluate preliminary total GAA protein, ATB200 and miglustat PK data and safety markers from 10 patients in this Phase 1/2 study.

[0208] This is a Phase 1/2, first-in-human, ascending-dose, fixed-sequence, open-label study to evaluate the safety, tolerability, PK, PD, and efficacy of intravenous infusions of ATB200 co-administered with oral miglustat in adults with Pompe disease (Figure 24). Mean total GAA protein and miglustat PK results from the first 8 patients in Cohort 1 through Visit 9 and the first 2 patients in Cohort 3 were assessed. aSafety data from 2 sentinel patients from Cohort 1 were reviewed at each dose level prior to dosing in Cohorts 2 and 3. bDuring Steps 2 and 3, miglustat was administered orally prior to initiation of the ATB200 intravenous infusion. For all doses, ATB200 was infused intravenously over a duration of 4 hours. cThe first 2 patients in Cohorts 2 and 3 served as sentinel patients for their respective cohorts. Key inclusion criteria: • Men and women aged 18–65 years diagnosed with Pompe disease based on documented deficiency of GAA enzyme activity or by GAA genotyping • Received ERT with alglucosidase alfa for 2–6 years (or >2 years for Cohort 2) prior to trial initiation (Cohort 1) • Currently receiving alglucosidase alfa at a frequency of every other week and completed the last 2 infusions without a drug-related adverse event resulting in dose interruption (Cohorts 1 and 2) • Must be able to walk 200 to 500 meters on the 6-Minute Walk Test (Cohorts 1 and 3) • Forced vertical vital capacity must be 30%–80% of predicted normal value (Cohorts 1 and 3). • Must be completely wheelchair bound and unable to walk unassisted (Cohort 2). PK analysis: • Blood samples for total plasma GAA protein and activity concentration were collected • Step 1: prior to start of ATB200 infusion and 1, 2, 3, 3.5, 4, 4.5, 5, 6, 8, 10, 12, and 24 hour(s) post-start of infusion • Steps 2 and 3: 1, 2, 3, 4, 4.5, 5, 6, 7, 9, 11, 13, and 25 hour(s) post-oral miglustat administration • Blood samples for plasma miglustat concentrations were drawn immediately prior to oral miglustat administration (time 0) and 1, 1.5, 2, 2.5, 3, 4, 5, 6, 9, 11, and 25 hour(s) after oral miglustat administration. Plasma miglustat is determined by a validated LC-MS/MS assay • Plasma total GAA protein concentrations for 5, 10, and 20 mg/kg ATB200 were determined by a validated LC-MS/mS quantification of rhGAA-specific “signature” peptide(s)

[0209] A preliminary analysis was completed on 8 patients in Cohort 1 who completed Steps 1 and 2 and 2 patients in Cohort 3 who started Step 3 • Patients with initial ERT switch are representative of the Pompe disease population, with a mean of 5.02 years on ERT (Table 6) Table 6: Baseline Characteristics an = 10 from Cohort 1 (outpatient ERT switch) via internal data analysis; n = 2 from Cohort 3 (no treatment). Total GAA Protein

[0210] When given alone, ATB200 increases in a slightly greater than dose-proportional manner (Table 7 and Figures 25A-25D). Variability appears to increase with miglustat dose (Figure 25C). Co-administration of 20 mg/kg ATB200 with high-dose miglustat (260 mg) increased total GAA protein exposure (AUC) by approximately 25% relative to ATB200 alone at 20 mg/kg. The distribution half-life (α phase) increased by 45%, suggesting that high-dose miglustat stabilizes ATB200 in plasma. An increase in distribution half-life is accompanied by an increase in AUC from time to maximum plasma concentration until approximately 12 hours post-dose. Increases in AUC and half-life can be seen in the log scale during the terminal elimination phase (Figure 25B). ATB200 demonstrated a relatively high volume of distribution. Total GAA protein disposition in plasma appeared similar between ERT-naïve (Cohort 3) and ERT-experienced (Cohort 1) patients (Figures 25A and 25D). Table 7: Total GAA Protein AUC = area under the curve; CLT = total body clearance; Cmax = maximum drug concentration; CV = coefficient of variability; MD = multiple dose; SD = single dose; ti/2 = half-life; tmax = time to maximum drug concentration; Vss = apparent volume of distribution at steady state. aGeometric mean (CV%). bMedian (min-max). cArithmetic mean (CV%). dn = 8. en = 7. fn = 2. Miglustat PK

[0211] Miglustat demonstrated dose-proportional kinetics (Table 8 and Figure 26). Plasma miglustat appears similar between single and multiple doses. Table 8: Miglustat PK Summary Pharmacodynamics

[0212] By visit 11 in ERT-experienced patients in Cohort 1 (Figures 27A and 27B): • Alanine aminotransferase (ALT) decreased in 5 of 8 patients; 4/4 patients with elevated baseline levels normalized • Aspartate aminotransferase (AST) decreased in 6 of 8 patients; 3/4 patients with elevated baseline levels normalized • Creatine phosphokinase (CPK) decreased in 6 of 8 patients; 2/6 patients with elevated baseline levels normalized • Urine glucose tetrasaccharide (HEX4) levels decreased in 8 of 8 patients

[0213] By week 4, all 4 marker levels decreased in the 2 patients in the untreated cohort (Cohort 3) (Figures 27C and 27D).

[0214] In Figures 27A-27D, data are represented as mean ± standard deviation.

[0215] Safety • No serious adverse events (AEs) or infusion-associated reactions were reported after 155+ total infusions in all patients • Treatment-emergent AEs, reported in 11/13 (84%) patients, were generally mild and transient. • Treatment-related AEs reported in 7/13 (53%) patients: nausea (n = 1), fatigue (n = 1), headache (n = 1), tremor (n = 2), acne (n = 1), tachycardia (n = 1), and hypotension (n = 1).

[0216] Conclusions • ATB200 alone and in combination with miglustat were safe and well tolerated, with no infusion-associated reactions to date. • ATB200 alone showed greater than dose-proportional increases in exposure, which were further enhanced with miglustat, suggesting a stabilizing chaperone effect of ATB200. • After switching from standard of care to ATB200/miglustat, patients generally showed improvement in biomarkers of muscle damage, with many patients demonstrating normalization by week 18. • The initial 2 treatment-naïve patients treated with ATB200/miglustat demonstrated robust reductions in all biomarkers of muscle damage

[0217] The embodiments described herein are intended to be illustrative of the present compositions and methods and are not intended to limit the scope of the present invention. Various modifications and changes consistent with the description as a whole and which are readily apparent to one skilled in the art are intended to be included. The appended claims are not to be limited by the specific embodiments set forth in the examples, but are to be given the broadest interpretation consistent with the description as a whole.

[0218] Patents, patent applications, publications, product descriptions, GenBank Accession Numbers, and protocols are cited throughout this application, the disclosures of which are incorporated herein by reference in their entirety for all purposes.

Claims (30)

1. A method for producing purified recombinant human acid alpha-glucosidase (rhGAA) comprising: in a bioreactor, culturing Chinese hamster ovary (CHO) host cells that secrete rhGAA; removing the medium from the bioreactor; filtering the medium to provide a filtrate; loading the filtrate onto an anion exchange chromatography (AEX) column to capture rhGAA; eluting a first protein product from the AEX column; loading the rhGAA eluted from the AEX column onto an immobilized metal ion affinity chromatography (IMAC) column; and eluting the rhGAA from the IMAC column, wherein the purified rhGAA comprises from 3.0 to 8.0 moles of sialic acid residues per mole of rhGAA and from 3.0 to 6.0 moles of M6P per mole of rhGAA, and comprises an amino acid sequence that is at least 98% identical to SEQ ID NO:2. 2. The method of claim 1, wherein the purified rhGAA comprises a first potential N-glycosylation site, a second potential N-glycosylation site, a third potential N-glycosylation site, a fourth potential N-glycosylation site, a fifth potential N-glycosylation site, a sixth potential N-glycosylation site, and a seventh potential N-glycosylation site. 3. The method of claim 1 or claim 2, further comprising: loading the rhGAA eluted from the IMAC column onto a third chromatography column; and eluting the rhGAA from the third chromatography column. 4. Method according to claim 3, characterized in that the third chromatography column is selected from a cation exchange chromatography (CEX) column and a size exclusion chromatography (SEC) column. 5. Method according to any one of claims 1 to 4, characterized in that the filtration of the medium is selected from alternating tangential flow filtration (ATF) and tangential flow filtration (TFF). 6. The method of any one of claims 1 to 5, further comprising inactivating viruses in one or more of the rhGAA eluted from the AEX column, the rhGAA eluted from the IMAC column, and the rhGAA eluted from the third chromatography column. 7. The method of claim 3, further comprising filtering the rhGAA eluted from the IMAC column or the rhGAA eluted from the third chromatography column to provide a filtered product. 8. The method of claim 7, further comprising lyophilizing the filtered product. 9. The method of any one of claims 1 to 8, wherein: (i) at least 90% of the purified rhGAA binds to the cation-independent mannose-6-phosphate receptor (CIMPR) or (ii) at least 90% of the purified rhGAA comprises an N-glycan carrying mono-mannose-6-phosphate (mono-M6P) or bis-mannose-6-phosphate (bis-M6P). 10. The method of any one of claims 1 to 9, wherein rhGAA comprises seven potential N-glycosylation sites located at amino acids corresponding to N84, N177, N334, N414, N596, N826, and N869 of SEQ ID NO: 2. 11. The method of any one of claims 1 to 10, wherein at least 3% of the total N-glycans in the purified rhGAA are bis-M6P. 12. The method of any one of claims 1 to 11, wherein at least 17% of the total N-glycans in the purified rhGAA are bis-M6P. 13. The method of any one of claims 1 to 12, wherein 3% to 25% of the total N-glycans in the purified rhGAA are bis-M6P. 14. The method of any one of claims 1 to 13, wherein 17% to 25% of the total N-glycans in the purified rhGAA are bis-M6P. 15. Method according to any one of claims 1 to 14, characterized in that the purified rhGAA comprises on average at least 1 mole of bis-M6P per mole of protein. 16. The method of any one of claims 1 to 15, wherein the purified rhGAA molecules comprise on average 1.3 mol of bis-M6P per mol of rhGAA. 17. The method of any one of claims 1 to 16, wherein at least 50% of the purified rhGAA molecules comprise a bis-M6P-containing N-glycan unit at the first potential N-glycosylation site. 18. The method of any one of claims 1 to 17, wherein at least 30% of the purified rhGAA molecules comprise a mono-M6P-containing N-glycan unit at the second potential N-glycosylation site. 19. The method of any one of claims 1 to 18, wherein at least 30% of the purified rhGAA molecules comprise a bis-M6P-containing N-glycan unit at the fourth potential N-glycosylation site. 20. The method of any one of claims 1 to 19, wherein at least 20% of the purified rhGAA molecules comprise a sialic acid-containing N-glycan unit at the third potential N-glycosylation site. 21. The method of any one of claims 1 to 20, wherein at least 20% of the purified rhGAA molecules comprise a mono-M6P-containing N-glycan unit at the fourth potential N-glycosylation site. 22. The method of any one of claims 1 to 21, wherein at least 50% of the purified rhGAA molecules comprise a sialic acid-containing N-glycan unit at the fifth potential N-glycosylation site. 23. The method of any one of claims 1 to 22, wherein at least 60% of the purified rhGAA molecules comprise a sialic acid-containing N-glycan unit at the sixth potential N-glycosylation site. 24. The method of any one of claims 1 to 23, wherein 30% to 65% of the purified rhGAA molecules are glycosylated at the seventh potential N-glycosylation site. 25. Purified recombinant human acid alpha-glucosidase (rhGAA) prepared by the method as defined in any one of claims 1 to 24. 26. Pharmaceutical composition characterized by the fact that it comprises purified rhGAA as defined in claim 25 and a pharmaceutically acceptable carrier. 27. Use of the pharmaceutical composition as defined in claim 26 or of the purified recombinant human acid alpha-glucosidase (rhGAA) as defined in claim 25 characterized by the fact that it is for the manufacture of a medicament for the treatment of Pompe Disease. 28. Use according to claim 27, characterized in that the medicament additionally comprises a pharmacological chaperone for acid α-glucosidase. 29. Use according to claim 28, characterized in that the pharmacological chaperone is selected from 1-deoxynojirimycin and N-butyl-deoxynojirimycin. 30. Use according to either of claims 28 or 29, characterized in that the pharmacological chaperone is coformulated with purified rhGAA.